Patent Application
20060188964
Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found at the end of the specification preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention claimed herein.
The guanine nucleotide-binding protein (“G-protein”) coupled receptor (“GPCR”) superfamily is one of the most diverse groups of proteins. GPCRs comprise membrane proteins involved in a wide range of physiological signaling processes, and are attractive targets for pharmacological intervention to modify these processes in normal and pathological states.
GPCRs activate signaling paths in response to stimuli such as Ca2+, amines, hormones, neurotransmitters, peptides (and even large proteins), chemokines, and sensory stimuli. For example, some GPCRs are involved in the receptors found in the tongue (i.e. affecting taste) and nose (affecting smell). Some GPCRs are involved in regulating heartbeat, and some GPCRs are opiate receptors in the brain which affect one's predisposition to drug addiction.
GPCRs share many structural features. For example, GPCRs share a transmembrane structural motif comprising seven a helices connected by six loops of varying lengths. Binding of specific ligands to the seven a-helical transmembrane domains of GPCRs causes conformational changes that act as a switch to signal a G-protein, which in turn evoke subsequent intracellular responses. Many studies have been conducted to develop an understanding of the precise conformational transformation of an inactive GPCR into an activated form capable of interacting with a G-protein, in order to elucidate the molecular steps of cell surface activated receptor-mediated intracellular signaling. However, a high-resolution visualization of the entire GPCR structure is needed to understand the mechanism of GPCR signal transduction.
For these reasons and others, it is desirable to develop an atomic-level understanding of transmembrane signal transduction by GPCRs. For example, there is a need for structures for receptors stabilized in various relevant states, as complexes with natural signaling ligands, with pharmacological agonists and antagonists, and with signaling partner proteins, notably heterotrimeric G ligand complexes. X-ray crystallography could in principle be used to determine structures of GPCRs at the desired resolution of detail.
However, determining the structure of a GPCR is a challenging task. Crystallization of GPCRs for X-ray diffraction studies is difficult because GPCRs are membrane proteins. In addition, most GPCRs are found naturally only in very small quantities. Purification of GPCR proteins from natural sources can be difficult and time consuming, and the amount of purified protein is often too small for structural studies and functional characterizations.
Many barriers remain for production, through conventional techniques, of membrane proteins at levels of abundance and quality suitable for structural determinations, and this is particularly true for eukaryotic proteins. All of the structures determined to date for eukaryotic membrane proteins have come from naturally abundant sources. An example of such abundant protein is bovine rhodopsin, which is the only GPCR whose structure has been resolved at the atomic level.
This invention provides a method for producing a membrane-bound protein in high yield, which comprises the steps of (a) culturing a mammalian cell and progeny thereof having therein an expression vector which coordinately expresses both (i) the membrane-bound protein and (ii) a luminescent protein, under conditions permitting selection of cells expressing the luminescent protein; (b) selecting cells cultured in step (a) which express a high yield of the luminescent protein so as to thereby select cells expressing a high yield of the membrane-bound protein; and (c) treating the cells selected in step (b) so as to recover therefrom the membrane-bound protein in high yield.
This invention also provides a first nucleic acid encoding a fusion protein comprising consecutive amino acids, the amino acid sequence of which corresponds to the amino acid sequence of a serotonin receptor and immediately contacting thereto the amino acid sequence of a targeting polypeptide which, upon expression of the fusion protein in a bacterium, causes the fusion protein so expressed to become situated in the bacterium's periplasmic space with the hydrophobic portion thereof being membrane-bound.
This invention further provides a second nucleic acid encoding a fusion protein comprising (a) a G protein coupled receptor (GPCR) and (b) a targeting polypeptide which, upon expression of the fusion protein in a bacterium, causes the fusion protein so expressed to become situated in the bacterium's periplasmic space with the hydrophobic portion thereof being membrane-bound.
This invention still further provides a third nucleic acid encoding a fusion protein comprising (i) a G protein coupled receptor (GPCR), (ii) a bacterial signal peptide, and (iii) a targeting polypeptide which, upon expression of the fusion protein in a bacterium, causes the fusion protein so expressed to become situated to the bacterium's periplasmic space with the hydrophobic portion thereof being membrane-bound.
This invention further provides a first, second and third bacterial expression vector comprising the first, second and third nucleic acid, respectively.
This invention further provides a method for producing a membrane-bound protein in high yield, which comprises the steps of (a) culturing a bacterial cell and progeny thereof having therein an expression vector which coordinately expresses both (i) the membrane-bound protein and (ii) a luminescent protein, under conditions permitting selection of cells expressing the luminescent protein; (b) selecting cells cultured in step (a) which express a high yield of the luminescent protein so as to thereby select cells expressing a high yield of the membrane-bound protein; and (c) treating the cells selected in step (b) so as to recover therefrom the membrane-bound protein in high yield.
This invention also provides a method for expressing a G protein coupled receptor (GPCR) in a bacterial cell comprising culturing a bacterial cell comprising the second or third expression vector.
This invention also provides a first fusion protein comprising serotonin receptor and a targeting protein which, upon the fusion protein's expression in a bacterium, causes the fusion protein to be directed to the bacterium's periplasmic space with the hydrophobic portion thereof remaining membrane-bound.
This invention further provides a second fusion protein comprising (a) a non-glycosylated G protein coupled receptor (GPCR) which binds to the ligand to which the glycosylated form of the GPCR binds, and (b) a targeting protein which, upon the fusion protein's expression in a bacterium, causes the fusion protein to be directed to the bacterium's periplasmic space with the hydrophobic portion thereof remaining membrane-bound.
This invention still further provides a third fusion protein comprising (i) a G protein coupled receptor (GPCR), (ii) a bacterial signal peptide, and (iii) a targeting protein which, upon the fusion protein's expression in a bacterium, causes the fusion protein to be directed to the bacterium's periplasmic space.
This invention provides a first, second and third bacterial cell comprising the first, second and third expression vector, respectively.
This invention also provides a method for determining which vector(s) among a plurality of G protein coupled receptor (GPCR)-encoding bacterial expression vectors give rise to a desired level of GPCR expression in bacteria comprising (a) culturing a plurality of populations of bacteria, wherein (i) each population is transfected with the second or third expression vector, (ii) each population of bacteria is comprised of the same strain as the others, and (iii) each population of bacteria is transfected with a different vector than are the other populations, and (b) determining which population(s) express the desired level of GPCR, thereby determining which expression vectors give rise to a desired level of GPCR expression.
This invention further provides a method for producing a bacterial spheroplast having a G protein coupled receptor (GPCR) affixed to the outer membrane thereof comprising (a) culturing the second or third bacterial cells, and (b) removing the outer cell membranes thereof.
This invention still further provides a bacterial spheroplast having a G protein coupled receptor (GPCR) affixed to its outer membrane.
This invention still further provides a method for determining whether an agent binds to a G protein coupled receptor (GPCR) comprising (a) contacting the agent with a bacterial spheroplast having the GPCR affixed to its outer membrane under conditions permitting binding of the GPCR on the spheroplast to a known ligand thereof, and (b) determining whether the agent binds to the GPCR on the spheroplast, thereby determining whether the agent binds to the GPCR.
This invention also provides a method for producing an antibody against a G protein coupled receptor (GPCR) comprising administering to a mammalian subject a bacterial spheroplast having the GPCR affixed to its outer membrane, so as to cause production in the subject of an antibody against the GPCR.
This invention also provides a method for identifying a reagent in which a membrane protein is likely to crystallize, which protein is known to be soluble in a detergent which preserves the protein's structural integrity, comprising the steps of (a) permitting equilibration between a solution containing the detergent at a predetermined concentration and a matrix of precipitant-containing reagents, wherein (i) the reagents collectively comprise a plurality of precipitant types and/or concentrations, and (ii) each reagent contains only one precipitant at one concentration; and (b) after equilibration occurs, identifying one of the equilibrated reagents, if any, in which cloud point has been achieved, such equilibrated reagent being one in which the membrane protein is likely to crystallize.
This invention also provides a method for producing crystals of a protein which, in a cell, is membrane-bound, comprising the steps of (a) identifying a reagent in which the membrane protein is likely to crystallize according to the instant method, and (b) growing crystals of the protein in the reagent identified in step (a).
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant mammalian cell-based method, and (b) treating the protein from step (a) so as to form crystals thereof.
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant mammalian cell-based method; and (b) treating the protein from step (a) so as to form crystals thereof, wherein the treating comprises the steps of
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant bacterial cell-based method; and (b) treating the protein from step (a) so as to form crystals thereof.
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant bacterial cell-based method; and (b) treating the protein from step (a) so as to form crystals thereof, wherein the treating comprises the steps of
This invention also provides a method for producing, and obtaining the crystal structure of, a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant mammalian cell-based method; (b) treating the protein from step (a) so as to form crystals thereof; and (c) obtaining a crystal structure for the crystals formed in step (b).
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant mammalian cell-based method; (b) treating the protein from step (a) so as to form crystals thereof, wherein the treating comprises the steps of
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant bacterial cell-based method; (b) treating the protein from step (a) so as to form crystals thereof; and (c) obtaining a crystal structure for the crystals formed in step (b).
Finally, this invention provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant bacterial cell-based method; (b) treating the protein from step (a) so as to form crystals thereof, wherein the treating comprises the steps of
FIGS. 14A and 14B: Ligand binding to membranes isolated from 293 cells enriched for the expression of serotonin-receptor. Saturation curve for tritiated LSD (
FIGS. 19A and 19B: Data from a typical experiment with expression of various MBP-5HT2c fusion constructs.
FIGS. 20A and 20B: Ligand binding to bacterial spheroplasts isolated from E. coli cells expressing the MBP-serotonin receptor fusion protein. Saturation curve for tritiated mesulergine (
FIGS. 21A and 21B: Expression data and relative activity data for the 5HT1a receptor.
FIGS. 23A and 23B: Results from testing different maltoside detergents which show yield and activity of 5HT2c receptor solubilized by maltoside detergents.
FIGS. 27A and 27B: Gel electrophoresis of purified MBP-receptor fusion protein. Each gel is stained by Coomassie blue to quantify protein. Denaturing polyacrylamide-SDS gel compared with molecular mass standards and bovine serum albumin (BSA) concentration standards (
FIGS. 28A and 28B: Silver-stained denaturing gel of fractions collected from the rerun of the 150 kDa species, and the corresponding activity profile, respectively.
FIGS. 29A and 29B: Expression and activity of C-terminal fusions to 5HT2c.
FIGS. 32A and 32B: Expression of olfactory receptor SP1.
Definitions
“Cloud point”, as used in the field of X-ray crystallography, means the precipitant concentration above which a soluble protein in solution becomes insoluble, and below which a soluble protein in solution remains soluble.
“Eukaryotic cell” means any cell with a true nucleus bounded by a nuclear envelope. Eukaryotic cells include, for example, animal cells (e.g., mammalian cells) and plant cells.
“Expression” means the cellular production of protein encoded by a particular nucleic acid. Expression includes, for example, transcription of DNA, processing of the resulting mRNA product and its translation into an active protein (see Sambrook et al. 1989).
“Expression vector” shall mean a nucleic acid encoding a nucleic acid of interest and/or a protein of interest, which nucleic acid, when placed in a cell, permits the expression of the nucleic acid or protein of interest. Expression vectors are well known in the art.
“Fusion protein” means a protein having a single polypeptide chain, which chain comprises two or more moieties which in nature do not exist as part of the same polypeptide chain. Examples of fusion proteins include a polypeptide chain comprising GPCR and MBP, wherein the GPCR and MBP are either contiguous or separated by a linker region.
“Functional GPCR” means an expression product which is effective as a receptor of the associated G-protein. As used herein, “GPCR” and “functional GPCR” are synonymous, unless otherwise indicated.
“GPCR” means G-protein coupled receptor. GPCRs include, without limitation, serotonin olfactory receptors, glycoprotein hormone receptors, chemokine receptors, adenosine receptors, biogenic amine receptors, melanocortin receptors, neuropeptide receptors, chemotactic receptors, somatostatin receptors, opioid receptors, melatonin receptors, calcitonin receptors, PTH/PTHrP receptors, glucagon receptors, secretin receptors, latrotoxin receptors, metabotropic glutamate receptors, calcium receptors, GABA-B receptors, pheromone receptors, and other G-protein coupled, seven-transmembrane segment receptors. In one embodiment, the GPCR has a loop deletion, or an N- and/or C-terminal truncation.
“GFP” means green fluorescent protein. GFP is a protein produced by the jellyfish Aequorea victoria which fluoresces bright green upon exposure to ultraviolet or blue light.
“Isolated” membrane-bound protein includes, for example, protein in isolated bilayers (either as naturally enriched or reconstituted), and in detergent micelles.
“Likely” to crystallize, with respect to a protein solubilized in a first reagent, means more likely to crystallize in the first reagent than in a second reagent.
“Luminescent protein” means any protein which gives off visible light upon exposure to ultraviolet or visible light (e.g., GFP).
“Mammalian cell” shall mean any mammalian cell. Mammalian cells include, without limitation, cells which are normal, abnormal and transformed, and are exemplified by neurons, epithelial cells, muscle cells, blood cells, immune cells, stem cells, osteocytes, endothelial cells and blast cells. Examples of mammalian cells commonly used for protein expression include HEK 293 cells, NIH 3T3 cells, CHO cells and TOF cells.
“Matrix” of reagents means a plurality of reagents in separate compartments. In one embodiment, the reagents are contained within a single apparatus. In another embodiment, the reagents are contained in a plurality of apparati. Apparati envisioned for this purpose include, without limitation, standard crystallization plates, and plates and scaffolds used in microassays and high-throughput screening.
“MBP” shall mean maltose-binding protein.
“Nucleic acid” shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., USA).
“PEG” means polyethylene glycol.
“PEG-related compound” means glycerol, ethylene glycol, or a derivative of PEG, such as PEG monomethyl ether or PEG dimethyl ether.
“Precipitant” means an agent which, at a high enough concentration, causes a solubilized protein to become insoluble. Precipitants include, for example, PEG, PEG-related compounds, salts, and small volatile organic compounds.
“Polypeptide” and “protein” are used equivalently, and each means a polymer of amino acid residues. The amino acid residues can be naturally occurring or chemical analogues thereof. Polypeptides and proteins can also include modifications such as glycosylation, lipid attachment, sulfation, hydroxylation, and ADP-ribosylation.
“Protein cleavage site” means a site recognized and cleaved by a site-specific protease, such as TEV protease which recognizes and cleaves the site ENLYFQ·GS.
“Stabilize”, with respect to a protein, means to inhibit the protein's degradation or any other physical modification which adversely affects its function.
The present disclosure describes methodologies for expressing, purifying, characterizing and crystallizing functional GPCRs.
Specifically, this invention provides a method for producing a membrane-bound protein in high yield, which comprises the steps of (a) culturing a mammalian cell and progeny thereof having therein an expression vector which coordinately expresses both (i) the membrane-bound protein and (ii) a luminescent protein, under conditions permitting selection of cells expressing the luminescent protein; (b) selecting cells cultured in step (a) which express a high yield of the luminescent protein so as to thereby select cells expressing a high yield of the membrane-bound protein; and (c) treating the cells selected in step (b) so as to recover therefrom the membrane-bound protein in high yield.
In one embodiment, the membrane-bound protein is a G protein coupled receptor (GPCR), such as a human GPCR. In another embodiment, the luminescent protein is green fluorescent protein (GFP). “Mammal” shall include, without limitation, a human, non-human primate, mouse, rat, guinea pig or rabbit.
In another embodiment, the instant method further comprises repeating steps (a) and (b) prior to step (c). In a further embodiment of the instant method, the vector further encodes a protein conferring resistance to an antibiotic, and the conditions permitting selection of cells expressing the luminescent protein encoded by the vector comprise the presence of the antibiotic in a medium in which the cells are cultured. “Antibiotic” includes, without limitation, ampicillin, kanamycin, chloramphenicol and tetracycline. In one embodiment of the instant method, the cells selected in step (b) have an average of at least 3 million copies of the membrane-bound protein per cell. In another embodiment, the cells selected in step (b) have an average of at least 5 million copies of the membrane-bound protein per cell. In a further embodiment, the cells selected in step (b) have an average of at least 10 million copies of the membrane-bound protein per cell.
This invention also provides a first nucleic acid encoding a fusion protein comprising a serotonin receptor and a targeting polypeptide which, upon expression of the fusion protein in a bacterium, causes the fusion protein so expressed to become situated in the bacterium's periplasmic space with the hydrophobic portion thereof being membrane-bound.
In one embodiment of the first nucleic acid, the serotonin receptor is human serotonin receptor. In another embodiment, the targeting polypeptide is maltose binding protein (MBP). In a further embodiment, the bacterium is E. coli. “Bacterium” includes, without limitation, E. coli and B. subtilis.
In another embodiment of the first nucleic acid, the fusion protein further comprises a first linker region between the serotonin receptor and the targeting polypeptide. In a further embodiment, the first linker region comprises a protein cleavage site and/or an affinity purification tag. “Affinity purification tag” includes, without limitation, biotin, poly-histidine, streptavidin-binding peptides and antibody tags.
In a further embodiment, the fusion protein further comprises a protein which stabilizes the serotonin receptor, such as Gα protein.
In a further embodiment of the first nucleic acid, the fusion protein further comprises a second linker region between the serotonin receptor and the polypeptide which stabilizes it. In one embodiment, the second linker region comprises a protein cleavage site.
This invention further provides a second nucleic acid encoding a fusion protein comprising (a) a G protein coupled receptor (GPCR) and (b) a targeting polypeptide which, upon expression of the fusion protein in a bacterium, causes the fusion protein so expressed to become situated in the bacterium's periplasmic space with the hydrophobic portion thereof being membrane-bound.
In one embodiment of the second nucleic acid, the GPCR is a human GPCR. In another embodiment, the targeting polypeptide is maltose binding protein (MBP). In a further embodiment, the bacterium is E. coli.
In another embodiment of the second nucleic acid, the fusion protein further comprises a first linker region between the GPCR and the targeting polypeptide. In one embodiment, the first linker region comprises a protein cleavage site and/or an affinity purification tag.
In a further embodiment of the second nucleic acid, the fusion protein further comprises a polypeptide which stabilizes the GPCR, such as Gα protein.
In a further embodiment of the second nucleic acid, the fusion protein further comprises a second linker region between the GPCR and the polypeptide which stabilizes it. In one embodiment, the second linker region comprises a protein cleavage site.
This invention still further provides a third nucleic acid encoding a fusion protein comprising (i) a G protein coupled receptor (GPCR), (ii) a bacterial signal peptide, and (iii) a targeting polypeptide which, upon expression of the fusion protein in a bacterium, causes the fusion protein so expressed to become situated to the bacterium's periplasmic space with the hydrophobic portion thereof being membrane-bound.
In one embodiment of the third nucleic acid, the GPCR is a human GPCR. In another embodiment, the targeting polypeptide is maltose binding protein (MBP). In a further embodiment, the bacterium is E. coli.
In another embodiment of the third nucleic acid, the fusion protein further comprises a first linker region between the GPCR and the targeting polypeptide. In one embodiment, the first linker region comprises a protein cleavage site and/or an affinity purification tag.
In a further embodiment of the third nucleic acid, the fusion protein further comprises a protein which stabilizes the GPCR, such as Gα protein. In another embodiment, the fusion protein further comprises a second linker region between the GPCR and the protein which stabilizes it. In one embodiment, the second linker region comprises a protein cleavage site.
In a further embodiment of the third nucleic acid, the fusion protein further comprises a third linker region between the signal peptide and the targeting polypeptide. In one embodiment, the third linker region comprises an affinity purification tag and/or a detection tag. “Detection tag” includes, without limitation, poly-histidine, an antibody, and a streptavidin-binding peptide.
This invention further provides a first, second and third bacterial expression vector comprising the first, second and third nucleic acid, respectively. In one embodiment, the vector is a vector for expression in E. coli.
This invention also provides a first method for expressing serotonin receptor in a bacterial cell comprising culturing a bacterial cell comprising the first expression vector. In one embodiment, the bacterial cell is E. coli.
This invention further provides a method for producing a membrane-bound protein in high yield, which comprises the steps of (a) culturing a bacterial cell and progeny thereof having therein an expression vector which coordinately expresses both (i) the membrane-bound protein and (ii) a luminescent protein, under conditions permitting selection of cells expressing the luminescent protein; (b) selecting cells cultured in step (a) which express a high yield of the luminescent protein so as to thereby select cells expressing a high yield of the membrane-bound protein; and (c) treating the cells selected in step (b) so as to recover therefrom the membrane-bound protein in high yield.
This invention also provides a second method for expressing a G protein coupled receptor (GPCR) in a bacterial cell comprising culturing a bacterial cell comprising the second or third expression vector. In one embodiment, the bacterial cell is E. coli. In another embodiment, the GPCR is a human GPCR.
This invention also provides a first fusion protein comprising serotonin receptor and a targeting protein which, upon the fusion protein's expression in a bacterium, causes the fusion protein to be directed to the bacterium's periplasmic space with the hydrophobic portion thereof remaining membrane-bound.
This invention further provides a second fusion protein comprising (a) a non-glycosylated G protein coupled receptor (GPCR) which binds to the ligand to which the glycosylated form of the GPCR binds, and (b) a targeting protein which, upon the fusion protein's expression in a bacterium, causes the fusion protein to be directed to the bacterium's periplasmic space with the hydrophobic portion thereof remaining membrane-bound.
This invention still further provides a third fusion protein comprising (i) a G protein coupled receptor (GPCR), (ii) a bacterial signal peptide, and (iii) a targeting protein which, upon the fusion protein's expression in a bacterium, causes the fusion protein to be directed to the bacterium's periplasmic space.
This invention provides a first, second and third bacterial cell comprising the first, second and third expression vector, respectively.
This invention also provides a method for determining which vector(s) among a plurality of G protein coupled receptor (GPCR)-encoding bacterial expression vectors give rise to a desired level of GPCR expression in bacteria comprising (a) culturing a plurality of populations of bacteria, wherein (i) each population is transfected with the second or third expression vector, (ii) each population of bacteria is comprised of the same strain as the others, and (iii) each population of bacteria is transfected with a different vector than are the other populations, and (b) determining which population(s) express the desired level of GPCR, thereby determining which expression vectors give rise to a desired level of GPCR expression.
This invention further provides a method for producing a bacterial spheroplast having a G protein coupled receptor (GPCR) affixed to the outer membrane thereof comprising (a) culturing the second or third bacterial cells, and (b) removing the outer cell membranes thereof.
This invention still further provides a bacterial spheroplast having a G protein coupled receptor (GPCR) affixed to its outer membrane. In one embodiment of the instant bacterial spheroplast, the spheroplast is an E. coli spheroplast. In another embodiment, the GPCR is a human GPCR.
This invention further provides a bacterial spheroplast produced by the instant method.
This invention still further provides a method for determining whether an agent binds to a G protein coupled receptor (GPCR) comprising (a) contacting the agent with a bacterial spheroplast having the GPCR affixed to its outer membrane under conditions permitting binding of the GPCR on the spheroplast to a known ligand thereof, and (b) determining whether the agent binds to the GPCR on the spheroplast, thereby determining whether the agent binds to the GPCR. In one embodiment, the agent is an antibody. “Antibody” includes, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, this term includes polyclonal and monoclonal antibodies, and fragments thereof (e.g., Fab fragments). Furthermore, this term includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.
This invention also provides a method for producing an antibody against a G protein coupled receptor (GPCR) comprising administering to a mammalian subject a bacterial spheroplast having the GPCR affixed to its outer membrane, so as to cause production in the subject of an antibody against the GPCR.
This invention also provides a method for identifying a reagent in which a membrane protein is likely to crystallize, which protein is known to be soluble in a detergent which preserves the protein's structural integrity, comprising the steps of: (a) permitting equilibration between a solution containing the detergent at a predetermined concentration and a matrix of precipitant-containing reagents, wherein (i) the reagents collectively comprise a plurality of precipitant types and/or concentrations, and (ii) each reagent contains only one precipitant at one concentration; and (b) after equilibration occurs, identifying one of the equilibrated reagents, if any, in which cloud point has been achieved, such equilibrated reagent being one in which the membrane protein is likely to crystallize. “Detergent”, when used in the context of protein crystallization, includes, without limitation, SDS, alkyl maltopyranosides, alkyl glucopyranosides, alkyl dimethylamine-N-oxides, digitonin, and alkyl FOS-CHOLINEs.
In one embodiment, the membrane protein is a GPCR. In another embodiment, the solution containing the detergent further comprises a buffer at a predetermined concentration. In a further embodiment, the precipitant in each reagent is selected from the group consisting of a PEG-related compound, a salt, an organic solvent, and a small volatile organic compound. “Salt” includes, without limitation, NaCl, an NH4+-containing salt, a Ca++-containing salt and a Mg++-containing salt. “Small volatile organic compound” includes, without limitation, MPD (2,4-methyl pentane diol), 1,6-hexane diol, and heptane triol.
In still a further embodiment, in step (a), the solution containing the detergent further comprises the membrane protein at a concentration and purity level suitable for crystallization, and in step (b) one of the equilibrated reagents, if any, in which protein precipitation has been achieved is identified, such equilibrated reagent being one in which the membrane protein is likely to crystallize. “Concentration and purity level” of protein sufficient for crystallization includes, for example, 0.5-40 mg/ml protein and 90-100% purity.
In another embodiment of this method, the method further comprises the steps of: (a) permitting equilibration between the equilibrated reagent identified in step (b) and a matrix of buffers, wherein (i) the buffers collectively have a plurality of pH's, and (ii) each buffer has only one pH; and (b) after a suitable period of time, identifying one of the equilibrated buffers, if any, in which protein precipitation or crystallization has been achieved, thereby identifying a reagent suitable for crystallizing the membrane protein. In a further embodiment, the method further comprises assessing the quality of any protein crystals formed.
This invention also provides a method for producing crystals of a protein which, in a cell, is membrane-bound, comprising the steps of (a) identifying a reagent in which the membrane protein is likely to crystallize according to the instant method, and (b) growing crystals of the protein in the reagent identified in step (a). In one embodiment, the protein is a G protein coupled receptor (GPCR).
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant mammalian cell-based method, and (b) treating the protein from step (a) so as to form crystals thereof.
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant mammalian cell-based method; and (b) treating the protein from step (a) so as to form crystals thereof, wherein the treating comprises the steps of
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant bacterial cell-based method; and (b) treating the protein from step (a) so as to form crystals thereof.
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant bacterial cell-based method; and (b) treating the protein from step (a) so as to form crystals thereof, wherein the treating comprises the steps of
This invention also provides a method for producing, and obtaining the crystal structure of, a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant mammalian cell-based method; (b) treating the protein from step (a) so as to form crystals thereof; and (c) obtaining a crystal structure for the crystals formed in step (b).
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant mammalian cell-based method; (b) treating the protein from step (a) so as to form crystals thereof, wherein the treating comprises the steps of
This invention also provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant bacterial cell-based method; (b) treating the protein from step (a) so as to form crystals thereof; and (c) obtaining a crystal structure for the crystals formed in step (b).
Finally, this invention provides a method for producing crystals of a protein which, in a cell, is a membrane-bound protein which comprises the steps of (a) producing the protein in high yield according to the instant bacterial cell-based method; (b) treating the protein from step (a) so as to form crystals thereof, wherein the treating comprises the steps of
To facilitate an understanding of the material which follows, one may refer to Sambrook et al. (1989) for certain commonly used methodologies and/or terms which are not described in detail herein.
The following Exemplary Embodiments and Experimental Details are set forth to aid in an understanding of the subject matter of this disclosure, but are not intended to, and should not be construed to, limit in any way the claims which follow thereafter.
Some embodiments are described exemplarily below to illustrate some methodologies contemplated by the subject disclosure. Many variations can be introduced on these embodiments without departing from the spirit of the disclosure or from the scope of the appended claims.
A method of amplified expression of a functional G-protein coupled receptor, according to one embodiment (
A method of amplified expression of a functional G-protein coupled receptor, according to another embodiment (
A method of amplified expression of a functional serotonin receptor, according to one embodiment (
A method of amplified expression of a functional 5HT2c receptor, according to one embodiment (
A method of amplified expression of a functional G-protein coupled receptor, according to another embodiment (
A method of amplified expression of a functional serotonin receptor, according to another embodiment (
For example, the cells that express the serotonin receptor may be isolated by cell sorting through flow cytometry. The cells that express the GFP may be isolated by hand selection of green fluorescent colonies. According to another embodiment, a puromycin-resistance marker also is inserted in the expression plasmid, and the eukaryotic cells, after transfection of the expression plasmid into the eukaryotic cells, is treated with puromycin to select transfected cells with stable integrated serotonin receptor. The isolated cells may further be cultured and sorted to obtain additional amplification. A cytomegalovirus promoter may be applied to the expression plasmid. Binding assays may be applied to verify that the isolated cells express the functional serotonin receptor.
The method may further comprise solubilizing the serotonin receptor in a detergent system to harvest the target functional serotonin receptor. The method also may further comprise inserting additives in the expression plasmid to preserve activity of the solubilized serotonin receptor.
Effective and general expression cloning methodologies for the expression of functional GPCR proteins into biological membranes are described herein, which may be applied to the production of, for example, functional serotonin receptors. Recombinant DNA are stably integrated into, according to one embodiment, cultured mammalian cells selected for highly amplified expression of relevant receptors. Appropriate expression plasmids may also be introduced into bacterial cells for high-level expression of function receptors.
Purification and biochemical characterization methodologies are described herein which may include detergent solubilization methodologies for isolating receptors from cell membranes and chromatographic separation procedures for purifying them. The functional state of the recombinant receptor molecules may be characterized by, for example, ligand binding measurements, both on cell membranes and as purified protein. Other biochemical properties of the purified receptors may also be analyzed.
Methodologies for production of receptor complexes stabilized with protein ligands are described herein, which may be used to prepare complexes of receptor molecules with protein ligands that can be expected to stabilize the proteins into a fixed conformation and to enhance the probability for crystallization by increasing the hydrophilic surface area. Candidates include cognate heterotrimeric G proteins and antigen-binding fragments from conformation-sensitive monoclonal antibodies.
In addition, methodologies for the crystallization and structure determination of GPCR and pertinent complexes are described herein.
Analysis of signal transduction mechanisms may use the obtained structural information to develop hypotheses regarding biophysical mechanisms for GPCR signal transduction. Such hypotheses may be tested through analysis of site-directed mutant variants, complexes with relevant ligands, and cellular assays of function.
Elements and/or features of different illustrative embodiments may be combined with each other and/or substituted for each other within the scope of this disclosure and appended claims. For example, in some additional embodiments, the purification methodologies described herein, unless specifically stated otherwise, may be applied to eukaryotic cell expression as well as to bacterial expression.
Some embodiments of the subject methodologies as applied experimentally to some G-protein coupled receptor are described exemplarily below. One experiment corresponds to expression of 5HT2c in one type of mammalian cell. Other experiments correspond to expression of 5HT2c and other GPCRs in one type of bacterial cell (Escherichia coli). The methodologies described herein (and other aspects of this disclosure) may be adapted for other G-protein coupled receptors (for example, 5HT2b, 5HT1c, other serotonin subtypes, other biogenic amine receptors, such as dopamine receptors, epinephrine receptors, norepinephrine receptors, histamine receptors, and neurotensin receptors), expression in other types of eukaryotic cells, and expression in other types of bacterial cells.
A cell perceives its environment through receptor molecules embedded in the plasma membrane and endowed with selective sensitivity toward various stimuli. Conformational changes or associations that occur when a receptor interacts with an external stimulus are transmitted to the cell interior where responses are induced, often elicited through a cascade of signal transduction events. The mechanism of signal transduction depends on molecular characteristics of the receptor. There are several classes of receptors. In addition to those linked to downstream elements by heterotrimeric G proteins, there are many receptors linked to protein tyrosine kinases, ones linked to ion channels, and diverse receptors coupled in other ways as in the TGFβ/Smad, Notch and Wnt systems.
The stimulus detected by a receptor may be physical (for example, light or an electrostatic potential), but in most cases the stimulus is a chemical ligand. Some ligands are macromolecules, and others are small compounds. Some are diffusible, and others are associated with another cell or the extracellular matrix.
The most salient molecular characteristic of G-protein coupled receptors (GPCRs) is a pattern of seven hydrophobic segments that correspond to transmembrane α-helices. Therefore, GPCRs are also known as seven transmembrane (7TM) receptors. This 7TM pattern was first seen in sequences of rhodopsins (Ovchinnikov et al. 1982; Hargrave et al. 1983; Nathans et al. 1983) and a little later in the sequence of the hamster β2-adrenergic receptor (Dixon et al. 1986). The involvement of heterotrimeric G proteins in signaling through 7TM receptors was first worked out for the β2-adrenergic receptor (Ross et al.), where binding of the hormone epinephrine activates Gas which in turn stimulates adenylyl cyclase production of the second messenger cyclic AMP. The parallel role of the G protein transducin in visual signaling, where photoactivation of rhodopsin stimulates cyclic GMP phosphodiesterase and sodium-channel closure, was discovered a little later (Stryer 1986). Taken in this context, the evident homology between these two biologically disparate 7TM receptors prompted the realization that they were the founding members of the GPCR family.
A flood of GPCR clonings ensued, including the first for serotonin receptors (Julius et al. 1988; Kobilka et al. 1987) and the discovery of the huge sub-family of odorant receptors (Buck et al. 1991). A total of 948 GPCR genes were identified in a recent analysis of the human genome sequence (Takeda et al. 2002), up somewhat from the 616 found initially (Venter et al. 2000). These receptors include sensors of exogenous stimuli, such as light and odors, and others that respond to endogenous ligands ranging from cationic amines such as serotonin, to peptides such as angiotensin, and further on to proteins such as chemokines and glycoprotein hormones.
Ligand binding (retinal photoisomerization in opsins) activates a GPCR to serve as a nucleotide exchange factor for the cognate heterotrimeric G protein. Each heterotrimer is a labile association of the GTPase component, Gα, and a Gβ:Gγ heterodimer. Gα(GDP):Gβ:Gγ dissociates to Gα(GTP) and Gβ:Gγ when stimulated by an activated GPCR, and the trimer reassociates after GTP hydrolysis. Both components are tethered to the membrane, by N-terminal myristolation or palmitoylation of Gα and C-terminal prenylation of Gγ, and after activation they can diffuse away from the receptor to effector sites on their membrane-associated targets.
There are at least 15 different Gα proteins, 5 Gβs and 5 Gγs (Conklin et al. 1993), and different combinations are selective for specific GPCRs and for target effector molecules (Gilman 1987). In particular, Gαs(GTP) stimulates adenylyl cyclase, whereas Gαi(GTP) inhibits it, and Gαq(GTP) stimulates phospholipase C-β. Crystal structures have been determined for Gα proteins in various states, including a complex between Gαs(GTP) and the catalytic portion of adenylyl cyclase, of Gβ:Gγ and of heterotrimers (Noel et al. 1993; Coleman et al. 1994; Wall et al. 1995; Lambright et al. 1996; Sondek et al. 1996; Tessmer et al. 1997).
GPCR receptors are thought to exist in equilibrium between inactive and active states, which naively correspond to empty and ligand occupied receptors. The ligand-binding site is known from studies on rhodopsin (Thomas et al. 1982) and on β2-adrenergic receptor (Strader et al.) to be located between helices near the center of the membrane. Conformational changes that accompany ligand binding or activation are linked to receptor binding of the G protein. G protein association with a receptor increases its ligand affinity.
A model of G-protein coupling of GPCR activation to effector targets is exemplified in
Many of the receptors that act in synaptic neurotransmission are GPCR family members. These include ATP and biogenic amines such as acetylcholine, dopamine, epinephrine, histamine and serotonin. Certain neuroactive peptides such as enkephalin and substance P can also act as neurotransmitters. These chemicals are synthesized in the pre-synaptic neuron, typically packaged into exocytic vesicles, and released into the synapic cleft upon neural excitation. They then diffuse across to the postsynaptic cell, typically another neuron or a muscle cell, where they can bind to cognate receptors. A specific presynaptic uptake transporter or an inactivating enzyme is usually used to remove the neurotransmitter from the site of action.
The importance of neurotransmission mediated by serotonin receptors is evident from the receptor pharmacology and tissue distribution in animal models (Barnes et al. 1999) and from pharmacological responses in humans (Kandel 2000). Serotonin receptors are a major site of action of certain mind-altering drugs, notably lysergic acid diethylamine (LSD). Depression can often be treated effectively with drugs that act on serotonergic pathways, notably by serotonin reuptake inhibitors such as fluoxetine (Prozac).
Mammals have at least fourteen structurally and pharmacologically distinct receptors for serotonin, 5-hydroxytryptamine (5HT), all but two of which are GPCR receptors. The 5HT3 exceptions are ligand-gated ion channels. The receptors are classified into seven evolutionary sub-families, members of which share common signaling linkages (Barnes et al. 1999), as shown in
The sequences of certain 5HT receptors are compared with other GPCRs in
The 5HT2c receptor (then called 5HT1c) was first cloned from rat (Julius et al. 1989) and found to have a predicted size of 460 amino-acid residues corresponding to a molecular mass of 51,899 Daltons. A schematic diagram of the amino-acid sequence and assignment of transmembrane segments is shown in
The characteristic 7TM pattern of hydrophobic segments in GPCRs provides powerful constraints on possibilities for 3D structure. Moreover, this pattern when seen in rhodopsin was reminiscent of that in bacteriorhodopsin where the structure from purple membranes had shown the disposition of helices [Henderson et al. 1977]. Although the sequences showed no detectable homology and these two photoreceptors have very different biochemical actions they do both use a Schiff-base linked retinal to detect light. The topological connections in bacteriorhodopsin were found at high resolution (Henderson et al. 1990). Electron crystallography also showed that the helices in rhodoposin are disposed generally as in bacteriorhodopsin. As sequences accumulated, conserved features were realized and comprehensive alignments were made (Probst 1992). The vast majority of GPCR sequences are homologous with rhodopsin, but there also are groups of the superfamily which are atypical. These include the secretin and metabotropic glutamate receptors.
GPCR sequence alignments reveal many features besides the 7TM pattern, some of which are evident in the subset shown in
Structural models have also been predicted from GPCR sequences (Zhang et al. 1993; Shacham et al. 2001). The best constrained model came from combining the structure of frog rhodopsin, determined at 9A resolution by electron microscopy of 2D crystals (Unger et al. 1997), with an analysis of the sequences of some 500 rhodopsin-family members (Baldwin et al. 1997). The result was an alpha-carbon template for the transmembrane helices for the rhodopsin family of GPCR receptors. Based on the Baldwin template, a rough three-dimensional model of the 5HT2C receptor was produced (
The natural abundance of rhodopsin in retinas makes this an exceptional GPCR protein. Most other GPCRs, including the serotonin receptor, are naturally scarce and therefore require the development of appropriate recombinant expression systems to support structural studies. Accordingly, methodologies for the production of recombinant serotonin receptor in sufficient yields for structural analysis and functional characterization were devised.
Integral membrane proteins present formidable, but not insurmountable problems for structural analysis. There have been striking successes starting with the first result in three dimensions, by electron crystallography at 7 Å resolution, on bacteriorhodopsin (Henderson et al. 1975) and the first atomic-level structure, at 3 Å resolution by x-ray crystallography, on a photosynthetic reaction center (Deisenhofer et al. 1985). Membrane-protein structures have been determined at an accelerated pace in recent years, and many of these new structures have had dramatic impact as in the cases of cytochrome c oxidases (Iwata et al. 1995; Tsukihara et al. 1996) and bacterial potassium and chloride channels (Doyle et al. 1998; Dutzler et al. 2002). Nevertheless, the structural output on membrane proteins is a very small fraction of that for soluble macromolecules. Through 1999, there were 12,896 PDB entries and 5348 novel macromolecular structures meeting the criteria for Macromolecular Structures (Hendrickson et al. 2000). The PDB total was nearly 19,000 in September 2002 (www.rcsb.org/pdb). Thus, while membrane proteins comprise 20-30% of all proteins in both prokaryotic and eukaryotic organisms (Wallin et al. 1998) they are but a fraction of a percent of those with known structure.
It is, of course, the natural association of membrane proteins with lipid bilayers that complicates their structural analysis. In the case of crystallography, for example, once suitably ordered crystals are obtained for a membrane protein, the diffraction analysis is as straightforward as it is for aqueous soluble macromolecules. By contrast, the biochemical preparation of pure membrane proteins is intrinsically much more challenging than for naturally soluble counterparts. One may isolate them in bilayers, either as naturally enriched or reconstituted, or make them water soluble in detergent micelles. Two-dimensional membrane arrays may be used for electron crystallography. There now are a few such atomic level structures. The membrane array also may be used for solid-state NMR experiments, and this technology is just coming of age. Soluble detergent micelles can be used for solution NMR experiments. New TROSY techniques are promising. Soluble detergent micelles also may be used for x-ray crystallography, which has dominated the field and is the approach of choice for the project proposed here. The crystallization of proteins in detergent micelles has its own special difficulties, including the following: (1) the protein may not be stable outside the lipid bilayer (Bowie 2001), (2) detergent interactions that occur during crystallization are important (Loll et al. 2001), and (3) the detergent-covered lipophilic surfaces are flexible and unavailable for lattice contacts (Ostermeier et al. 1997), which theoretically reduces the probability of crystallization by a high power of the fractional surface area (Kwong et al. 1999).
Problems that arise in the recombinant expression of membrane proteins are even more limiting than difficulties in purification and crystallization. There have been recent successes in producing recombinant bacterial proteins for analysis. Eukaryotic membrane proteins have been strikingly recalcitrant in expression at the scale needed for structural analysis. Although there are structures of important eukaryotic membrane proteins, they have all come from natural sources except for the peripheral, single-leaflet associated cyclooxygenases (Picot et al. 1994). Many mammalian membrane proteins of interest, including the serotonin receptor, are scarce and cannot be prepared from natural membranes as for retinal rhodopsin (Okada et al. 2000). Moreover, GPCR systems do not exist in prokaryotes and thus bacterial homologs, exploited so effectively in potassium channel studies (Doyle et al. 1998), are not an option in this case. Appropriate recombinant expression systems are therefore needed to support such structural studies. Various expression systems are being adapted for the production of mammalian membrane proteins (Grisshammer et al. 1995).
A study of the structural biology of G-protein coupled receptors (GPCRs) may be multi-faceted. One dimension may include the study of, for example, the neurobiology of olfaction and neurotransmission. It has been found, for example, that the perception of smell begins with a large class of specific GPCRs, the olfactory receptors (Buck et al. 1991). Another facet may include studying structural aspects of signal transduction, such as analyzing structures of several molecules involved in signaling through protein tyrosine kinases, which may include the following: protein ligands such as fibroblast growth factor (FGF) (DiGabriele et al. 1998), stem cell factor (Jiang et al. 2000), and ciliary neurotrophic factor (CNTF) (McDonald et al. 1995); tyrosine-kinase portions from the insulin receptor (Hubbard et al. 1994) and lymphocyte kinase (Yamaguchi et al. 1996); and ligand-binding portions from an FGF receptor (Stauber 2000) and the angiogenesis receptor Tie-2. In addition, other structural studies have been conducted on CD4 (Ryu et al. 1990; Ryu et al. 1994; Wu et al. 1997; Wu et al. 1996) and CD8 (Leahy et al. 1992), which are tyrosine-kinase linked components of the cellular immune response. Signaling through the histidine-kinase receptors of two-component systems has also been studied, and the structure of the histidine-kinase portion of PhoQ (Marina et al. 2001) and a number of the sensor domains from such receptors were determined.
Serotonin receptor 5HT2c is a suitable entry point for structural efforts on GPCRs for the following reasons: (1) there is a body of work on the neurobiology of this and related serotonin receptors, (2) a mouse fibroblast cell line which is available has been shown to express the receptor at a high level (Julius et al. 1989), and (3) there are well characterized ligands for this receptor. Methodologies for amplified expression in mammalian cells and in bacterial cells as a fusion protein are described herein. Recombinant receptors expressed in either membrane system are shown to have wild-type activity. Additional methodologies are described herein which may be used to solubilize, purify and crystallize the receptor molecules from both systems. Methodologies established in the 5HT2c studies have been adapted for the expression of 5HT1a, 5HT1b and 5HT7. Structural studies on other GPCRs, including olfactory receptor, glycoprotein hormone receptors, and chemokine receptors used by HIV, have also been undertaken and are discussed below.
Amplified Expression of Functional 5HT2c in Mammalian Cells
Expression of the rat 5HT2c serotonin receptor in the initial mouse fibroblast cell line, NIH 3T3, was at a level of 103 to 104 high-affinity binding sites for 125I-labelled LSD per cell (Julius et al. 1988). The subsequent tumor-cell lines showed enhanced expressions levels of up to 8×105 receptors per cell (Julius et al. 1989), which compares favorably to the estimated natural level of approximately 105 receptors per cell of the choroid plexus. Production of mammalian proteins for crystallography in Chinese hampster ovary (CHO) cells was successful in connection with extracellular portions of the T-cell coreceptors CD4 and CD8. An expression plasmid coupled to dihydrofolate reductase (DHFR) and driven by the cytomegalovirus (CMV) promoter was devised to produce CD8 (Leahy et al. 1992). Upon transfection into DHFR-deficient CHO cells, those cells exhibiting multi-copy integration at optimal sites are expected to show reduced susceptibility to the DHFR inhibitor, methotrexate. Selection for methotrexate resistance was then used to amplify CD8 expression. Attempts were made to improve upon 5HT2c expression in 3T3 cells. In one approach, tumor-derived mouse fibroblast cell lines expressing the 5HT2c receptor were generated using the protocol established by Julius et al. Through multiple rounds of flow cytometry, cells were selected on their ability to respond to progressively decreasing concentrations of agonist. Cells were permeabilized with a calcium sensitive dye and the cellular response was monitored by fluorescent detection of the calcium released from the intracellular stores. Expression levels were monitored at each cycle of sorting and selection. Expression levels gradually increased to approximately 1×106 molecules/cells.
In a second approach, a nine-residue epitope specifically recognized by an anti-hemagglutinin (HA) monoclonal antibody was fused to the N-terminus of the 5HT2c receptor. It was hoped to select high-expressing cells by labeling the cells with the anti-HA antibody which could be monitored in flow cytometry with a fluorescent secondary antibody. Fluorescent intensity of a given cell was expected to correlate with the amount of receptor expressed on its surface. Unfortunately any modification to the N-terminus of the 5HT2c receptor abolished expression.
Greater success was obtained with expression in 293 human embryonic kidney (HEK) cell lines. The amplification system devised for expression of the serotonin receptor in HEK293 cells employs green fluorescent protein (GFP) as a selectable marker (
GFP can be used as a selectable marker for expression in mammalian cells. GFP offers two main advantages: the intensity of fluorescence correlates with the expression levels of the foreign gene, and the highest expressing cells can be selected by FACS sorting and amplified. The expression level of a transfected and selected cell line is stable over time. Expression of GFP is correlated to the expression of the gene of interest. However, since translation is independent for GFP and the target protein, fluorescence of the former does not assure proper targeting and folding of the latter in the cell membrane. An antibody epitope may be genetically fused to the N-terminus of different serotonin receptors that are unlikely to possess a signal sequence. This tag can be used to assess expression. Once stable integrants have been selected by antibiotic resistance, a flow cytometry experiment similar to that performed with GFP can be devised. Antibodies can be fluorescently labeled and can be used for selection and amplification of transfected cells. The advantage of this approach is that fluorescence correlates directly to the number of antigen sites (and therefore receptors) on the cell surface.
A plasmid was developed to include genes for both GFP and 5HT2c as well as a puromycin-resistance marker (step S111). The puromycin-resistance marker is under control of its own promoter, and the 5HT2c and GFP genes, separated by an internal ribosome entry site (IRES), are under the control of the same strong CMV promoter used previously. Stable integrants were selected by treating transfected cells with puromycin (steps S112 and S113). Cells that had productively and repeatedly integrated GFP typically also produce the receptor in similarly high abundance (step S114). Cells that express GFP highly can be isolated readily either by hand picking of green fluorescent colonies or by cell sorting via flow cytometry (step S115). Selected cells can then be cultured and sorted again for further amplification (step S116).
The results of GFP selection are shown in
Binding assays were performed in order to verify that the selected cell cultures did express functional serotonin receptors and to quantify expression levels. Tritiated LSD was bound to membranes isolated from enriched cells through saturation (
The data show that the ligand bound with Kd ˜1.0 nM (as seen in vivo) and that these membranes expressed ˜140 pmoles of functional receptor per mg of membrane protein. On a per cell basis, this corresponds to ˜1.5×106 LSD-binding sites per cell, and these cell lines do not diminish over time. This level of expression is sufficient, at least in principle, to support structural studies. Four ten-layer cell farm runs suffice to produce ˜500 mg of total membrane protein which corresponds to 3.5 mg of functional receptor in the cell membrane.
Tags of various nature suitable for purification of the receptor were genetically fused to its C-terminus without hindering expression levels or activity. Once effective expression protocols were established and the products could be purified, characterized and set up for crystallization (see discussion below), attempts were made to remove potential sources of conformational heterogeneity that might interfere with crystallization. The glycosylation pattern of the 5HT2c receptor expressed in mammalian cells was analyzed. Western blot analysis of cells harvested from a stable 293 cell line expressing the 5HT2c receptor was performed.
Each asparagine residue at a potential glycosylation site on 5HT2c was mutated to aspartic acid. Constructs were generated to express each single mutant individually (N39D, N204D and N205D), as the three possible combinations of double mutants, and as the triple mutant. These constructs, together with the expression plasmid encoding the wild type receptor were transfected into 293 cells. The cells were harvested 48 hours after transfection. Each population of cells was divided into two, one of which was deglycosylated by treatment with endoglycosidase F. Products were analyzed by western blot (
Surprisingly, expression levels of the 5HT2c receptor varied considerably amongst the different constructs. The single and to some extent also the double mutants seemed to express less than the triple mutant. The expression level of the triple mutant appeared comparable to that of wild type receptor. Overall expression levels were low, making it possible to detect only the non-glycosylated or deglycosylated form of the receptor. Nevertheless, one can conclude from this experiment that all three potential sites are indeed glycosylated. One can also conclude that a non-glycosylated form of the receptor can be expressed at levels comparable to that of the wild-type protein.
A stable cell line expressing the triple-mutant form of the receptor which cannot be glycosylated was generated using GFP as a marker. The three most fluorescent clones that could be selected by flow cytometry were amplified and tested for expression levels.
Cell lines were also generated in which the conserved, and most probably palmitoylated, cysteine after the seventh transmembrane domain (C387) was mutated to either alanine or serine. Neither the expression levels nor ligand-binding profiles were affected in these mutant receptors.
Expression of Functional 5HT2c as Fusion Protein in E. coli
There are advantages and disadvantages to alternative expression systems. The expression of a mammalian receptor in mammalian cells offers the advantage of presenting the expressed gene with the proper translational and membrane-insertion machinery for functional translocation to the cell surface. On the other hand, mammalian derived cell lines require great expense and technical expertise. In addition, the generation of highly expressing cell lines is troublesome and time consuming. This limits the number of mutant variants that can be examined in a reasonable amount of time. Prokaryotic expression has great advantages. Bacteria are relatively simply to grow, easy to scale, and most importantly, the time between genetic construction of a target and its expression as protein is short. Moreover, structural analyses might benefit from having molecules that lack post-translational modifications such as glycosylation and palmitoylation, which can negatively affect the formation of highly ordered crystals. This potential advantage can be a detrimental disadvantage if such post-translational modifications are required for correct folding and function of the protein to be expressed. Work on the 5HT2c receptor expressed in mammalian cells showed that neither glycosylation nor palmitoylation are essential requisites for this receptor. The major caveat with bacterial expression of eukaryotic membrane proteins is whether receptors can indeed be functionally inserted into the plasma membrane of a bacterial cell.
Initial attempts to express functional 5HT2c receptor in E. coli centered on the observation that the processed N-terminus of the receptor expressed in 293 cells actually begins at Ile33 rather than at the start of translation. This datum was shown by N-terminal sequencing of purified protein expressed in 293 cells. The presence of a signal sequence in the 5HT2c receptor had been hypothesized but never unambiguously demonstrated (Abramowski et al. 1995). It was thought that expression could be achieved by substituting the endogenous, previously unidentified signal sequence, with one of bacterial origin. Several different bacterial signal sequences were tried, including the signal sequences for the outer membrane protein gene ompT, and for the periplasmic and inner membrane protein genes malE, lacY and pelB. These experiments resulted in only low levels of expression, although some specific activity was observed. No specific activity was detected when the receptor was expressed with its endogenous signal sequence nor in the absence of any signal sequence.
Next, another method of amplified expression of a functional G-protein coupled receptor was developed (
A rationale for this methodology is that the translation and membrane translocation machinery of the bacterial cell is ‘primed’ by the bacterial leader protein and that the heterologous receptor protein follows in course to be inserted into the membrane. An expression system for the neurotensin receptor has been described (Grisshammer et al. 1993). Functional expression of a GPCR as a fusion to MBP has been shown to work for the β-adrenergic receptor (Hampe et al. 2000), the A2 adenosine receptor (Weiss et al. 2002), and the M2 muscarinic receptor (Furukawa et al. 2000).
Extensive optimizations of the expression conditions were performed. The variables screened included the bacterial strain, the media used for bacterial growth, the strength of the promoter, the IPTG inducer concentrations and the temperature of incubation.
Several secretion vectors were engineered for ready interchange of receptor constructs. Maltose-binding protein (MBP) fusions were used in each of these, but various other signal sequences were tried. Fusions were constructed with thrombin-cleavable linkages of various lengths; and several short, non-cleavable linkages were also made. Based on N-terminal sequencing of receptor purified from HEK 293 expression of 5HT2c, the processed N-terminus actually begins at Ile33 rather than at the start of translation. The MBP side of the shortened junctions was defined by the degree of ordering (B factors) in the crystal structure, which ends in a C-terminal helix.
The choice of the construct appeared to affect expression levels considerably. The length of the linker between MBP and the receptor was varied, and a recognition site for thrombin was inserted (step S182). Short linkers seemed to express better than long ones.
Surprisingly the C-terminal fusion of the bacterial cytoplasmic protein thioredoxin (TRX) to residue 402 of the receptor (15 residues after the palmitoylated cysteine 387) increased expression levels substantially (step S183). This amplification does not occur if TRX is fused to the C-terminus of the receptor, although TRX fused to the C-terminus of the neurotensin receptor has been shown to promote an increase in expression levels (Tucker et al. 1996). Ultimately, the expression was increased from an initial yield of ˜30 μg per liter of culture to between 500 μg and 1 mg per liter.
Different constructs and different variables that were screened were compared for resulting expression by running a fixed amount of cells on a gel and performing western blot analysis. Specific relative activity was assayed by converting an equal number of cells to spheroplasts and screening at a fixed concentration of radioligand. Data from a typical experiment with expression of various MBP-5HT2c fusion constructs is shown in
Once conditions were established for expression at a high level, the specific activity of the expressed protein was fully analyzed on an absolute scale, as for material from mammalian cells. In this case, experiments were carried out on spheroplasts (bacterial cells stripped of their outer membranes) using tritiated mesulergine (
Expression tests in E. coli can be performed rapidly since the rate-limiting step is construction of the expression plasmid. Expression cassettes were engineered in such a way that a gene can be inserted into any of several expression vectors with a single cloning step (step S184). The MBP-receptor fusion may be extracted from membrane preparations by applying the one or more detergents (step S185), as discussed below. With the thrombin recognition sequence inserted between the receptor and the MBP, the MBP can be removed quantitatively by adding thrombin (step S186).
Expression of 5HT1a, 5HT1b and 5HT7 as Fusion Proteins
Expression of three other serotonin receptors (human 5HT1A, murine 5HT1B and murine 5HT7) was investigated by genetically engineering fusions of these proteins to MBP. Different constructs were generated for each protein, and the site of fusion to MBP was varied along the N-terminus of the receptor. Some fusions were made close to the N-terminus, some close to the first transmembrane segment, and others in between the two. Experience derived from work on the 5HT2c receptor was used as a guideline in designing these constructs.
Expression data and relative activity data for the 5HT1a receptor are shown in
Preparation of 5HT2c Receptors from Mammalian Cells
HEK 293 cells expressing the 5HT2c receptor were harvested and lysed osmotically. The nuclear fraction together with unlysed cells and cellular debris were pelleted by mild centrifugation (<1000×g). Crude membranes were then harvested by ultracentrifugation. Over 50 different detergents were screened and tested for their ability to extract the 5HT2c receptor in a functional form from the isolated membranes. Typically, the experiments were performed as follows.
Membranes were resuspended at a concentration of membrane protein of approximately 10 mg/ml. Radioligand was added. The membranes were then diluted two fold with buffer (control), and with different detergents at 2% minimum concentration. A higher concentration was used for those detergents that have a critical micellar concentration (CMC) of above 2%. After one hour incubation at 4° C., the detergent insoluble fraction was separated from the soluble fraction by ultracentrifugation at 100,000×g for one hour. The extraction efficiency of a given detergent was tested by quantitative western blot analysis. The activity of the solubilized fraction was assayed using small size exclusion columns to separate the bound from the free radioligand and compared to activity measurements performed on non-detergent treated membranes.
Results from such an experiment, in this case testing different maltoside detergents, are shown in
A deca-histidine tag was genetically engineered to the C-terminus of the receptor for purification. This tag was fused to the wild type receptor as well as to the mutant form of the receptor that cannot be glycosylated or palmitoylated (discussed above). Cell membranes were washed at both low and high ionic strength to eliminate peripherally attached membrane proteins and other contaminants. The solubilized receptor was purified by metal-affinity chromatography. Digitonin and FOS-choline 12 were the detergents used in this experiment. The purified protein could be concentrated to approximately 3-4 mg/ml.
Analysis of the purified 5HT2c receptor is shown in
The ability of the purified material to bind ligand was assayed by radioligand-soluble binding assays. Traces of radioligand were also added to the membranes and fractions at different stages of the purification assayed for activity. Gel filtration experiments were also performed with traces of radioligand. Other purification schemes were also investigated, including ion-exchange chromatography and immunoaffinity chromatography based on an antibody epitope fused to the receptor C-terminus.
Preparation of 5HT2c Receptors from Bacterial Cells
The MBP-5HT2c receptor fusion was extracted from membrane preparations by screening the solubilization efficacy of various detergents. Following similar protocols to those for mammalian membranes, efficiency of solubilization was monitored by western blot analysis on denaturing gels, and activity was tested by soluble ligand binding assays. It was noted that the set of detergents suitable for the receptor varied between mammalian and bacterial membranes, presumably because of differences in membrane composition. In general, the bacterially-expressed receptor was found to be less stable than the one expressed in mammalian cells, again probably because of differences in lipidic composition in the two membranes. In order to stabilize the 5HT2c receptor produced in E. coli, additives were screened for their ability to preserve activity of the solubilized species. The most striking example found was cholesteryl hemisuccinate (CHS). CHS has been used successfully as a detergent additive for the solubilization and purification of other GPCRs (Weiss et al. 2002; Furukawa et al. 2000; Mirzabekov et al. 1999; Grisshammer et al. 1999).
A thrombin recognition sequence was engineered between MBP and the 5HT2c receptor. MBP could be removed quantitatively by the addition of thrombin. The cleaved receptor (i.e. lacking the N-terminal MBP) was shown to bind ligand as effectively as does the uncleaved fusion protein.
Three constructs were also generated lacking a linker between MBP and 5HT2c. MBP, as shown by its crystal structure (Spurlino et al. 1991), terminates in an alpha helix. In these three uncleavable constructs, the receptor was fused to each of the last three structurally ordered residues of the C-terminal helix of MBP. The objective was to sample different MBP-receptor relative orientations in order to facilitate crystallization. These constructs expressed as well and were as active as those carrying a linker, and they were resistant to thrombin proteolysis. Cleavage of the endogenous MBP signal peptide was shown to occur by N-terminal sequencing of the purified material.
The MBP portion of the fusion was shown to be functional by binding of the solubilized complex to amylose resin. However, amylose affinity-chromatography could not be used for purification in the presence of maltoside detergents since the carbohydrate moiety of these detergents interferes with binding of MBP to the resin.
A deca-histidine tag was genetically fused to the C-terminus of the receptor preceded by a thrombin cleavage site, and purification was achieved by metal affinity chromatography. Substantial purification was also achieved prior to solubilization by isolating the bacterial membranes to remove cytosolic proteins. These membranes were subsequently washed with a sub-solubilizing concentration of detergent to eliminate many peripherally attached membrane proteins.
The sample, after metal affinity chromatography, was characterized further in a variety of ways including activity assays and mass spectrometry. Mass spectrometry confirmed that the C-terminally fused deca-histidine tag could be proteolytically removed. Gel filtration was also used to improve purity and to characterize the oligomeric state of the protein. The protein was found to migrate as a broad 300 kDa and as a sharp 150 kDa protein. Both fractions were stable and bound ligand specifically as could be assayed by the gel-filtration profile of each fraction purified in a first run and run individually in a second run.
The purification protocols rely on affinity tags engineered to the C-terminus of the 5HT2c receptor. A deca-histidine tag preceded by a thrombin recognition site has proven to be the most successful. C-terminal modifications do however pose some concerns. The C-termini of GPCRs are extremely diverse in both amino acid composition and length. A tag might be accessible to its target affinity matrix for one receptor and not for another. The presence of a C-terminal tag might provoke unpredictable instability to a fusion partner. Expression levels for the bacterially expressed neurotensin receptor have been shown to vary according to the nature of C-terminally-fused affinity tag (Tucker et al.). In addition, proteases (such as thrombin) cleave after their substrate recognition sequence, leaving behind several typically unstructured residues which remain on the N-terminal side, and therefore on the protein, after proteolysis. These additional residues could have a detrimental effect in a crystallization experiment.
The purification scheme preferably allows for efficient purification of any receptor independent of its identity. Affinity chromatography of the bacterial fusion protein by binding of MBP to an amylose resin has been used to great success. The genetic engineering of affinity tags in the link region between MBP and the receptor, on the MBP side of the protease site (so that the tag does not remain attached to the protein after proteolysis) may be further investigated. Additional experiments may focus on polyhistidine tags and streptagII (Shinzawa-Itoh 1995), because of the difficulties often encountered in eluting an antigen-tagged protein from an antibody column. Both efficiency of purification and efficiency of proteolytic cleavage of receptor from MBP may be tested for each construct.
A more interesting approach involves the placement of affinity tag within the body of maltose binding protein. MBP is highly tolerant to multiple amino acid deletions and insertions in several positions (Duplay et al. 1987). These sites have been mapped and extensively characterized (Betton et al. 1993). Two positions, one at residue 133 and the other at 303, are particularly interesting because they are entirely solvent accessible and are located at a considerable distance from the C-terminus of MBP, where the receptor is fused. Antigen epitopes inserted at these loci have shown efficient recognition by the corresponding antibodies. MBP mutants with several different affinity tags inserted at these positions in MBP may be generated. Given the distance of these regions from the site of the fusion between MBP and the receptor, antibody binding to the internal antigen tags may be compatible with specific proteolysis of the engineered protease sites between MBP and the receptor, allowing elution of the unfused receptor from the antibody resin.
The reconstitution of detergent-solubilized protein into the lipid bilayers, typically in liposomes, can be used to verify structural integrity and to screen for any lipids optimal for activity and perhaps for crystallization (Cerione et al. 1983). Lipids have proved critical to the formation of many highly ordered crystals of membrane proteins, and reconstitution provides an excellent way to determine which lipid or class of lipids promotes maximal activity of the receptor. Receptor purified in the detergent system described herein for the bacterial system (dodecyl maltoside+cholesteryl hemisuccinate) can be used to reconstitute in vesicles made from a variety of natural lipid sources (e.g. E. coli, bovine brain, egg) and then tested by radioactive ligand binding assay for activity. Using the reconstitution assay the detergent screen can be repeated to discover any additional detergents missed during the initial screen which could extract receptor in a functional state. Some detergents cause structural perturbations to the receptor which can drastically alter ligand affinity. Nevertheless, they may sufficiently preserve structure enough to allow physiological activity to be recovered following lipid reconstitution and detergent removal. Such detergents may also be amenable to crystallization and provide meaningful structural data about the protein.
Reconstitution could also afford an excellent way to concentrate and purify the receptor. Following reconstitution, protein could then be extracted in a pure state from the bilayer at various detergent:lipid concentrations to find the right balance between the solubility and structural integrity suitable for crystallization. The eukaryotic membrane protein structures which have been solved to date have come from the direct detergent solubilization of protein from naturally abundant and pure sources (Shinzawa-Itoh et al. 1995; Toyoshima et al. 2000; Palczewski et al. 2000). An authentic reconstitution membrane could simulate this natural environment.
Receptor Complexes Stabilized with Protein Ligands
Proteins that interact with other molecules usually are stabilized by the interaction. Crystal structures of complexes often show lower flexibility than those for corresponding apo states, for example. In reverse, it can be expected that membrane proteins may lose stability when removed from their natural lipid bilayer environment into a detergent miscelle. There is a large entropic penalty for the formation of crystal contacts involving flexible regions. Thus, such regions tend to be excluded from contact and crystallization probability is sharply reduced when a substantial portion of the molecular surface is flexible (Kwong et al. 1999). In the case of solubilized membrane proteins, where substantial fractions of the surface are inherently flexible, this becomes an important factor. A strategy for using antibody fragments in membrane protein crystallization has been expounded (Hunte et al. 2002) and has been applied with good effect (Iwata et al. 1995; Ostermeier et al. 1997; Zhou et al. 2001). Another example comes from attempts to crystallize HIV gp120 (Kwong et al. 1999). Crystals were obtained when flexible loops were removed to make core constructs, deglycosylated to remove further conformational heterogeneity, and stabilized the protein with CD4 and antibody ligands.
A polyclonal antibody raised against a synthetic peptide corresponding to the extracellular 4-5 loop of the olfactory receptor SP1 has been shown to recognize this receptor by immuno-staining and immuno-precipitation under non-denaturing conditions from solubilized nasal tissue. A peptide corresponding to the same region of 5HT2c has been synthesized. As a first step towards the production of monoclonal antibodies, mice have been immunized with the SP1 and 5HT2c peptides. Serum collected from mice immunized with SP1 peptide has been shown to immuno-precipitate SP1 expressed in E. coli (details of the bacterial expression are discussed below). Two glycosylation sites are in the extracellular 4-5 loop of both rat and mouse 5HT2c. Therefore, it is possible that the corresponding (unglycosylated) peptide may elicit an immune response. Following a similar reasoning, if one or more monoclonal antibodies are obtained in this way they may be useful only for 5HT2c expressed in bacteria.
Expression Stabilized by Fusions with Gα and Other Partners
In vivo the 5HT2c receptor interacts with Gαq. The last 11-amino acid residues of the alpha subunit of the G-protein heterotrimer are known to be critical for the interaction with its cognate receptors (Martin et al. 1996). A chimeric Gα construct, called GαiqC, was made in which the C-terminal 11 residues of Gαi were replaced with those of Gαq. This protein was engineered because functional Gαi can be expressed in bacteria and Gαi has proven to be more structurally tractable than Gαq. The 5HT2c receptor was genetically fused to these two Gα subunits. Fusions were made to the full length receptor and also, as in the robustly expressing fusions of 5HT2c to TRX, to 5HT2c truncated at residue 402. Twenty different constructs were generated, in which 5 linkers were tried for each of the 4 combinations of receptor and Gα. These MBP-5HT2c-Gα fusions were expressed in E. coli following the same protocol used for the MBP-5HT2c constructs. Regarding expression and relative specific activity of MBP-5HT2c-Gαq and MBP-5HT2c-GαiqC fusions in comparison to those of MBP-5HT2c and MBP-5HT2c(402)-TRX, and as observed before, expression levels seem to correlate rather well with activity data. Although these fusions do not express as well as MBP-5HT2c(402)-TRX some express substantially better than MBP-5HT2c. The length of the linker seems to be an important factor. Moreover, comparison of the intensity of the 5HT2c degradation band between different constructs shows that in some cases the presence of the Gα subunit is protecting if not indeed stabilizing the receptor.
There is a rich literature on GPCR-GA fusions (Seifert et al. 1999). A series of Gα fusions to the 5HT2c receptor have been generated and tested, and a library of fusions between a series of serotonin receptors and their cognate G proteins is in production. These receptor-Gα pairs are 5HT2c-Gαq (which were also emulated with GiαqC), 5HT1a-Gαi, and 5HT7-Gas. Five linkers of different lengths and composition are being tested for each of the pairs in assays of expression and ligand-binding activity. Experiments are in the context of the MBP fusions for expression. Comparisons can be made to the well characterized expression pattern for the MBP-5HT and the MBP-5HT2c(402)-TRX fusions. Besides the aim of achieving stabilization, these or follow-up experiments with other serotonin sub-family members (
In view of evidence that one or more of the fusion constructs are functional they may be screened for suitable extraction conditions. Different detergents as well as stabilizing additives, such as lipids and cholesterol derivatives, can be screened, and extraction efficiency and ligand-binding activity can be assayed. Functionality of the detergent-solubilized species can be further assessed by probing its ability to interact with the G dimer. The β1γ2 isoform of the dimer can be produced in baculovirus-infected insect cells following well established protocols (Ueda et al. 1994). This isoform shows broad promiscuity in its interaction with different G subunits. The G-protein heterotrimer can be stably docked to the activated cognate GPCR when nucleotide free, and eluted by addition of GTP (Brown et al. 1993; Knezevic et al. 1993; Santos-Alvarez et al. 2000).
The formation of the quaternary complex (receptor-G fusion and Gβdimer) can be assayed initially by size-exclusion chromatography and native-gel electrophoresis. One could also test complex assembly by a co-immunoprecipitation experiment wherein one of the components (either the receptor-Gα fusion or Gβγ dimer) is bound to an inert support matrix (Pang et al. 1989). If complex formation is successful, the components co-elute from the column. In the case of GPCRs, specific dissociation of the purified complex can be achieved by addition of receptor agonist and GTP. Functionality of both partners in the fusion is paramount. By assaying the incorporation of radiolabelled GTPγs (a non-hydolyzable analogue of GTP), upon addition of agonist (serotonin) in the presence of Gβγ it can be determined if both receptor and G-protein are functional (Pang et al. 1989; Wurch et al. 2001). An ultimate test of functionality is to generate a proteolytically cleavable receptor-Gα fusion and to determine whether the quaternary complex reassembles with Gαβγ after protease treatment. Size-exclusion chromatography may be the most efficient way to address this question.
Various efforts may be made to raise monoclonal antibodies against conformational epitopes. A parallel effort has been made with a synthetic constrained peptide corresponding to an external loop from the 5HT2c receptor. This peptide may elicit an immunogenic-response, and the resulting monoclonal antibody-producing cells can be screened by non-denaturing ELISA (Padan et al. 1998) and immunoprecipitation. Alternative approaches to antibody generation include immunizing mice with detergent-solubilized purified protein, with protein that has been reconstituted in a lipid bilayer, with bacterial spheroplasts, or cells.
Crystallization of 5HT2c Produced in Mammalian Cells
Crystallization experiments were performed with the material purified as described above. Commercially-available screens as well as in-house screens were tested (see description below). Different temperatures were screened, and crystallization trials set up at 4° C. were found to yield the most promising results. Different ligands were also screened for their ability to promote crystallization. Mesulergine and serotonin were amongst the ligands screened. Polyethylene glycol precipitants on average gave better results than salt-based crystallization conditions. So far only small (<10 μM) crystals have been obtained. Although too small to perform meaningful diffraction experiments, pools of crystals were isolated, washed, run on a denaturing gel and western blotted. These experiments suggested that, at least for some conditions, the crystals did contain 5HT2c receptor.
Crystallization of 5HT2c Fusion Protein
Uncleavable fusions of MBP and 5HT2c prepared as described above were used for crystallization experiments. The majority of these experiments were performed using in-house designed screens. The design of the crystallization screens began with the assumption that the behavior of protein-detergent complexes is driven primarily by the properties of the detergent micelles alone. Detergents exhibit a property known as the “cloud point”, at which they undergo a phase transition that partitions the detergent into two separate detergent-rich and detergent-poor phases. If detergent-protein complexes are approximately like detergent micelles, then the location of this detergent-only phase transition in n-dimensional crystallization space should be located near the boundary of crystallization for the detergent-solubilized membrane protein.
A set of potentially useful detergents (several maltosides and several glycosides) initially were screened, at a given useful concentration above the critical micellar concentration (CMC), against a series of precipitants at increasing concentrations. From this simple mono-dimensional screen, the concentration at which a given precipitant-detergent combination exhibits phase separation can be estimated. To the positives from the initial screen (which are referred herein as “PF1”) several other variables such as pH buffers and a series of common secondary precipitants at different concentrations were added to generate PF2, the second generation screen. It was assumed that the addition of buffers and other precipitants at relatively low concentrations would not drastically shift the phase transition boundary of the detergent. Purified, detergent-solubilized protein was then used at this stage to determine the initial effectiveness of this approach. Against this second generation screen, an encouraging number of conditions yielded protein precipitates.
The hits from the second screen were then expanded and the concentrations of components were systematically screened more finely to optimize the crystallization parameters. The character of the protein precipitate improved with each generation screen. Iteration of this process eventually led to a sixth generation crystallization screen (PF6) which yielded protein crystals of promising morphology (FIG. V) which were subject to x-ray diffraction analysis (FIG. W). The rationale behind this approach, that solubilized membrane proteins should crystallize near the phase transition boundary of their detergent components, has also been used with considerable success by others (Loll et al. 2001; Song et al. 1998; Garavito et al. 1996).
The discussion above of crystallizing bacterially expressed 5HT2c receptor centers on constructs in which MBP and the receptor are fused directly without a proteolytically cleavable linker. Bacterially expressed material may include proteins in which the MBP has been cleaved prior to crystallization. Constructs in which MBP and 5HT2c are separated by a proteolytically-cleavable linker have already been generated and tested for expression, activity and efficiency of cleavage.
Receptor complexes with stabilizing ligands have been produced, and have an enhanced probability for crystallization. The majority of membrane protein structures solved to date have demonstrated a specific lipid requirement and in many of the structures, the electron density of a specific lipid molecule has been observed (Valiyaveetil et al. 2002). The lipid requirements of the 5HT2c receptor for crystallization can be investigated by extracting the purified receptor from reconstituted lipid bilayers made using a variety of individual lipids and lipid mixtures. Extraction can be done with various detergents at different concentrations to determine the optimal detergent:lipid ratio. Crystallization may also be attempted in cubo. Some lipids naturally form three-dimensionally ordered structures which can be used as a platform to induce crystallographic contacts of membrane proteins incorporated into these lipid structures. These cubic lipidic phases have been used to successfully crystallize a variety of retinal-conjugated bacterial membrane proteins (Landau 1996).
Crystals that are grown can be tested for diffraction quality by exposure to x-ray beams, such as at the synchrotron facilities at Brookhaven and Argonne National Laboratories. The testing may focus appropriately cryo-preserved samples, but capillary mounts may also be used to test intrinsic diffraction quality. It may be desired to obtain crystals that diffract sufficiently well to permit the construction of atomic-level models, which means diffraction at least as far as 3.5 Å and preferably better. Given similarities with rhodopsin, it may be possible to use the method of molecular replacement for structure determination, but experimental phases from the methods of multiple isomorphous replacement (MIR) or multiwavelength anomalous diffraction (MAD) may prove advantageous. In this regard, the bacterial expression system provides the now routine possibility for incorporation of selenomethionine for MAD analysis (Hendrickson et al. 1990; Hendrickson 1991).
The methodologies described herein can be extended to other members of the serotonin GPCR family as well as other GPCRs.
Olfactory Receptors
Olfactory receptors represent the most abundant class of GPCRs. These proteins are particularly resilient to expression in eukaryotic cells. The absence of identifiable high-affinity ligands for the majority of these proteins also poses a disadvantage in attempting to characterize these molecules biochemically and structurally. Nevertheless, the expression of an olfactory receptor was attempted as a fusion to MBP in bacteria. A particular receptor of murine origin named SP1 was chosen, a choice based mainly on the availability of a specific antibody. In an approach similar to the one followed for the expression of serotonin receptors, MBP was genetically fused to three different positions along the N-terminus of this receptor. Similar expression protocols to those used to express serotonin receptors were followed.
In order to further assess the quality of the expressed fusion protein, bacterial membranes were generated. The receptor was found to be associated entirely with the membrane fraction.
Glycoprotein Hormone Receptors
Existing studies have generated a longstanding interest in glycoprotein hormone receptors, having determined the structure of human chorionic gonadotropin (hCG) (Kwong et al. 1998) and made an analysis of hormone binding to the large, extracellular leucine-rich repeat domains of these receptors (Jiang et al. 1995). The model produced from the studies suggests that a loop from the hormone α-chain may interact with the transmembrane GPCR domain to activate the receptor. Complexes of hCG with the extracellular portion of human leuteinizing hormone receptor (LHR) and also of follicle stimulating hormone (FSH) complexed with the extracellular domain of its FSH receptor have been produced. Crystals have been grown in each case, and efforts are ongoing to improve protein supplies and diffraction characteristics of the crystals.
Chemokine Receptors Co-Opted by HIV
Studies on the interaction of the external envelope glycoprotein with its cellular receptors have yielded structures of gp120 complexed with the D1D2 domain of CD4 and the Fab portion of a neutralizing antibody that interacts with chemokine co-receptors. The structures include complexes of both a laboratory-adapted strain (Kwong et al. 1998), which interacts preferentially with the CXCR4 chemokine receptor, and also a primary isolate (Kwong et al. 1998), which interacts with CCR5. Procedures for the enhanced expression of CCR5 in mammalian cells (Mirzabekov et al. 1999) have been developed.
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Introduction
High-resolution structural studies of proteins generally require large amounts of pure, properly folded material. Indeed, the advent of gene manipulation techniques for producing recombinant protein in heterologous systems is arguably the most important breakthrough of the last thirty years for structural biology, exceeding even the wonderous developments in synchrotron crystallography [1] and NMR spectroscopy [2]. Bacterial expression systems, primarily based on the gram negative bacterium Escherichia coli, have been by far the most successful for the production of recombinant proteins for structural studies. Of the 14,011 protein structures deposited in the Protein Data Bank with ‘Expression_System’ records in their PDB entries (www.rcsb.org), 90.6% were produced using bacterial hosts. This success arises from several factors including the ease with which such organisms can be genetically manipulated; the thorough understanding of their transcription and translation machinery, which has led to the ability to achieve high levels of protein expression; the rapidity of their growth; and the relatively low cost of their use. Despite these advantages, bacterial systems often fail in their application to the expression of eukaryotic proteins [3, 4]. Failure to achieve acceptable expression often arises from toxicity of the foreign protein or its inability to fold or be targeted properly in the bacterial cell. Such problems inevitably result in low levels of expression or protein mis-folding [3, 4]. Thus, despite drawbacks in efficiency, alternative expression systems based on eukaryotic hosts, have been developed for large scale protein production. These include expression in yeast, insect cells, and mammalian cells.
Expression of mammalian proteins has proven to be particularly challenging in heterologous non-mammalian cell systems. It is difficult to ascribe specific reasons for lower levels of success found for mammalian proteins. However since mammalian proteins evolved in the milieu of mammalian cells, it is understandable that both proper folding and stability depends on their presence in this environment. Although it is routine to use mammalian protein expression systems in functional studies, their application to large scale protein production has been deterred by difficulty in obtaining large amounts of material, rapidly, and at a reasonable cost.
Transfection of mammalian cells leads to cell populations heterogeneous with respect to the amount of protein produced by each cell. Initial heterogeneity primarily arises from differences in the number of plasmids entering each cell in the stage of transient expression [5]. Protein expression levels in transiently transfected mammalian cells peak around 48-72 hours after transfection, and inevitably decline thereafter. Thus, the production of stable transfectants is desirable as a constant source of recombinant protein. Generation of stably producing cell lines requires integration of the expression construct into the genome of the host cell. This leads to additional sources of heterogeneity in expression levels, arising from differences in the number of integrants, and their sites of integration.
Here we describe a system for the selection of highly expressing stable mammalian cells, based on the detection of a co-expressed visible marker, the green fluorescent protein (GFP) [6, 7]. GFP provides an excellent means for cytologically localizing a product from any foreign gene which is fused to the GFP. The fused protein is typically functional and can be localized to its site in the cell through its green fluorescence. GFP has the advantage that it is a clonable marker for use in living tissue. In addition, unlike other bioluminescent reporters, it does not require additional proteins, substrates or co-factors to emit light.
In the GFP system, the coding sequence for the gene of interest is placed under the control of a strong constitutive promoter (such as the promoter element derived from cytomegalovirus, CMV [8]). Downstream, after the termination codon for the gene of interest, an internal ribosome entry site (IRES) [9] is followed by the coding sequence for GFP. Transcription from this construct produces a single bicistronic messenger RNA encoding both genes. The IRES element enables binding of the ribosome at the initiation site of GFP. Thus, two separate proteins—the gene of interest and GFP—are translated from the same message, and expression levels of both proteins are thereby coupled.
This system enables efficient selection of high expressors by monitoring the fluorescence of GFP. Use of fluorescence-activated cell sorting (FACS) [10, 11] technology allows for rapid selection of either clonal or non-clonal populations of highly expressing cells.
Methods and Results
As a test for the applicability of this system to high-level expression of functional proteins, we chose two targets, each presenting different challenges. The first target is the rat serotonin receptor subtype 2c (5HT2c) [12], a G-protein coupled receptor (GPCR). GPCRs are a large family of integral membrane proteins characterized by seven transmembrane spanning helices. GPCRs are notoriously resistant to structural studies, in part due to the difficulty of attaining high-level expression of functional protein [13, 14]. To date only one GPCR, bovine rhodopsin, has yielded a high-resolution structure [15, 16]. Rhodopsin, unlike other GPCRs, is present at high levels in rod cell outer segments where it is naturally expressed. The crystal structure of rhodopsin was determined using material purified from natural sources, rather than with a recombinant expression system. The second target, mouse resistin, is a highly disulfide-linked hormone that is naturally secreted from adipocytes [17, 18]. Attempts at expression of resistin in E. coli, either as soluble protein or refolded from inclusion bodies, does not yield properly folded functional protein [19, 20]. Resistin adopts a complex multimeric structure [21], which inevitably represents a challenge to reproduce with fidelity in heterologous expression hosts.
Expression of 5HT2c
The cDNA for the rat 5HT2c, which encodes a protein of 460 amino acids with three potential glycosylation sites and one palmitoylation site, was inserted into the multiple cloning site of the pCMV-IRES-GFP vector (pFM1.2,
Expression of Resistin
For expression of mouse resistin we inserted its coding sequence into pFM1.1 (
Discussion
The method described here enables the rapid generation of high-expressing stable mammalian cell lines. The entire procedure, from transfection to obtaining the final cell line, can be accomplished in less than two months time. While this is slow in comparison to bacterial expression methods, it is comparable to the time scale of other widely used methods such as infection of insect cells with recombinant baculovirus, generation of yeast stable integrants, or the production of mammalian cell lines using traditional techniques.
The GFP selection method provides significant advantages in comparison to conventional methods of cell line generation. The isolation of stable integrants in mammalian cells is generally accomplished with the use of antibiotic markers. Expression levels amongst these antibiotic-resistant colonies are highly variable. Traditionally, to screen for high expressing cells, individual colonies are hand-picked, and assayed for their levels of protein production by biochemical methods, usually involving immunological detection. These procedures are time consuming and labor intensive, and thus only a limited number of colonies can be screened. In contrast, the GFP-based selection method described here provides an efficient means for identifying and isolating highly-expressing cells.
The ideal marker for highly expressing cells would be the protein of interest itself. For example, to isolate high expressors of a fluorescent protein one would simply monitor the natural fluorescence. However, for most proteins, no detection method is available. Direct linkages between a protein of interest and a fluorescent marker can easily be constructed as gene fusions, but these are not generally suitable for structural studies. The separation of the fluorescent marker from the protein of interest through the use of an IRES element, enables the production of unmodified protein suitable for structural studies, while maintaining the correlation between expression level and fluorescence within each cell. Furthermore, this separation of target and marker renders the system generally applicable to expression of any protein.
Isolation of the most highly fluorescent cells, corresponding to the highest expressors, can be accomplished in a number of ways. First, visual inspection of fields of colonies enables rapid identification of the most suitable candidates, which can be manually isolated. Alternatively, colonies can be pooled and subjected to FACS analysis. Although, as shown here, the generation of clonal cell lines is not a requirement for achieving high-level expression, the current generation of cell sorters do allow for single cell cloning. Thus, clonal cell lines can be produced with equivalent ease.
Other fluorescent markers, most notably conjugated antibodies, can also be used to select highly expressing cells [23]. Although such markers provide direct correlation to protein expression levels, their use is limited to membrane-attached proteins with extracellular epitopes. Conjugated antibodies cannot enter the cell without a prior lethal permeabilization step, nor are they of use for secreted proteins which are no longer attached to the cells. In contrast, although the GFP selection method correlates fluorescence with expression at the mRNA level, it is not restricted to a limited class of proteins, nor does it depend on the availability of fluorescent markers that bind the protein of interest.
The GFP selection system has potential for future enhancements. These include the possibility for co-expression of multiple genes by constructing bicistronic messages for each, with a different fluorescent protein such as YFP or CFP (Clontech, Inc). This can enable sorting at multiple wavelengths in order to select cells that express all of the proteins highly. Toxic proteins can often be tolerated by cells only under tightly controlled inducible expression. Inducible mammalian expression systems have recently become widely available, and have proven extremely valuable for high level expression of proteins that negatively impact cell viability [24, 25]. Although the system described here provides constitutive expression, in principle it can be modified to provide inducible expression by changing the promoter element.
As structural biology progresses towards the elucidation of increasingly complex macromolecular structures, so too will the need for abundant supplies of the appropriately assembled recombinant molecules. Inevitably, this will lead to an increasing dependence on mammalian expression systems. The GFP selection method presented here addresses one critical step, that of the identification and isolation of highly expressing cells.
1. Hendrickson, W. A. (2000). Synchrotron crystallography. Trends Biochem Sci 25, 637-643.
This application claims the benefit of U.S. Provisional Application No. 60/592,056 filed Jul. 28, 2004, the contents of which are hereby incorporated by reference.
This invention was made with funding from the National Institutes of Health under grant numbers GM68671, GM68671, GM62529, R01-DK55758 and T32-GM97288. Accordingly, the United States Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60592056 | Jul 2004 | US |
