Patent Application
20230349847
The Instant Application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Apr. 26, 2023 is named “ZYP0281US2” and is 3,268 bytes in size.
The present application relates to the technical field of structural biology, and more particularly to a method for determining a protein structure using cryo-electron microscopy.
The statements herein only provide background information related to the present application, and do not necessarily constitute prior art.
High-resolution three-dimensional structures of proteins deliver important information for understanding the function of these proteins (Garcia-Nafria et al, 2020). Classically protein structures are determined using X-ray crystallography and NMR spectroscopy. Depending on the sort of protein, different hurdles have to be overcome to yield a high-resolution protein structure. In the case of X-ray crystallography properly diffracting crystals have to be produced requiring large quantities of purified protein. In the case of NMR spectroscopy, the protein structure can be investigated in solution but has to be labelled in the backbone and side chains with suitable isotopes. Both structure determination techniques are very time-consuming and can be extremely expensive. This is especially the case for determining the three-dimensional structure of membrane proteins (Li et al, 2021). About one third of the human proteome are membrane proteins. Many of them play a central role for the proper function or the dysfunction of cells. Their misfunction is responsible for many serious diseases and therefore membrane proteins are the targets for most of the modern medicines. Their importance for modern drug development will increase further (Sriram and Insel, 2018).
For the determination of the three-dimensional structure of membrane proteins additional hurdles do exist as compared to water-soluble proteins. Membrane proteins first have to be isolated out of their native cellular membranes, which is typically done by dissolving the entire membrane in detergents in order to yield membrane proteins in a detergent-solubilized molecular state. Under these conditions, membrane proteins can be purified in homogenous form. For X-ray crystallography, the detergent-solubilized membrane protein has to be crystallized. In most cases membrane proteins only yield highly diffracting crystals after severe modification of their native sequence. Structure determination of membrane proteins by NMR spectroscopy are faced by additional restrictions of the size of the proteins: Only the structure of proteins of a molecular mass below 80 kDa can be routinely solved with presently available technology (Purslow et al, 2020).
Recent developments in single-particle imaging using cryo-electron microscopy (cryo-EM) have revolutionized membrane protein structural biology (Külhlbrandt, 2014; Garcia-Nafria et al, 2020). Here, many individual membrane proteins or membrane protein complexes in detergent-solubilized form or reconstituted into lipid bilayer nano-discs are imaged. From the images of millions of individual particles, it is possible to estimate the 3D structure at atomic resolution of the corresponding wild-type membrane proteins, i.e. without the need of a crystal.
Cryo-EM structures beyond 2 Å resolution have been obtained for a few large or symmetric complexes (Greber et al, 2021). However, many drug targets are neither large nor symmetric. Presently, the wider application of single-particle cryo-EM for improved resolution of membrane protein structures have been restricted by the following factors:
In view of the above-described problems, it is an objective of the present application to provide a method for determining a protein structure using cryo-electron microscopy, which is a generic method that can solve the structure determination of target proteins of molecular mass down to at least 20 kDa.
To achieve the above objective, in accordance with one embodiment of the present application, there is provided a method for determining a protein structure using cryo-electron microscopy. The method comprises:
The scaffold protein is any one of streptavidin, avidin, or derivatives thereof. The tag is configured for selectively binding to the scaffold protein. The tag is one selected from the group consisting of: a biotin tag, comprising a biotin; a biotinylated protein or polypeptide tag, comprising a protein sequence and a biotin covalently linked to the protein sequence; a Strep-tag; and a biotinylated or strep-tagged antibody, or antibody Fab fragment, or single-chain antibody.
In an embodiment, the Strep-tag is a polypeptide sequence adapted to selectively bind with streptavidin or streptactin or other derivatives thereof.
In an embodiment of the present application, in case that the tag is the biotin tag, the step of enabling the target protein to contain the tag comprises:
In an embodiment, the biotin tag is site-selectively attached to the target protein via chemical modification or genetic engineering.
In an embodiment of the present application, the biotinylated protein or polypeptide tag is one selected from the group consisting of:
In an embodiment of the present application, the ACP-tag or the PCP-tag and the biotin are covalently attached enzymatically by Sfp- and AcpS-PPTases, respectively, using biotin derivates of CoA as substrates.
In an embodiment of the present application, the self-labeling protein tag is a Snap-tag, a Clip-tag, or a Halo-tag.
In an embodiment of the present application, the target protein has a molecular mass preferentially between 50 kDa and 20 kDa or less. In an embodiment of the present application, the target protein has a molecular mass down to at least 33 kDa. In an embodiment of the present application, the target protein has a molecular mass down to at least 20 kDa.
In an embodiment of the present application, the target protein is a water-soluble protein or a membrane protein.
In an embodiment of the present application, in the case that the target protein is a G protein-coupled receptor (GPCR), the biotin tag or the biotinylated protein or polypeptide tag is inserted to the N-terminus, the C-terminus, one of extracellular loops (that is, E1, E2, or E3), or one of intracellular loops (that is, I1, I2, I3, or I4) of the GPCR.
In an embodiment of the present application, in the case that the target protein is a G protein-coupled receptor (GPCR), an anticalin or an Ig type or single-chain antibody is selectively bound to the N-terminus, the C-terminus, one of extracellular loops (E1, E2, or E3), or one of intracellular loops (I1, I2, I3, or I4) of the GPCR.
In an embodiment of the present application, the biotin tag or the biotinylated protein or polypeptide tag or a Strep-tag is fused into an intracellular loop I4 of the GPCR, so as to stabilize a bound streptavidin or streptactin in a rigid structure.
In an embodiment of the present application, the biotin tag or the biotinylated protein or polypeptide tag or the Strep-tag is fused into one of extracellular loops (E1, E2, or E3) or one of intracellular loops (I1, I2, I3, or I4) of the GPCR, so as to stabilize a bound streptavidin or streptactin in a rigid structure.
In an embodiment of the present application, in the case that the target protein is a complex of a prototypical GPCR and an intracellular signaling protein, the tag is introduced by adopting any one of the following manners, so as to enable binding of the complex to the streptavidin or the derivative thereof as the scaffold protein:
In an embodiment of the present application, the intracellular signaling protein is any one selected from the following:
In an embodiment of the present application, in the case that the target protein is a neurokinin 1 receptor (NK1R), the tag is a biotinylated Halo-tag, the scaffold protein is streptavidin; a tetrameric NK1R is assembled on the streptavidin via the biotinylated Halo-tag, whereby forming a complex SA(HaloTag-NK1R)n, in which, n is an integer ranged between 1 and 4, for example, n is 1, or 2, or 3, or 4. The complex SA(HaloTag-NK1R)n is suitable for determining a structure of the NK1R by the cryo-EM.
In an embodiment of the present application, the biotinylated Halo-tag is inserted into a third intracellular loop IL3 of the NK1R, and a sequence of between amino acid 227 and 237 is removed from the third intracellular loop.
In an embodiment of the present application, before the step of enabling the target protein to contain the tag, the method further comprises:
In an embodiment of the present application, the HaloTag-PEG4-biotin ligand has a molecular formula of C31H57ClN4O9S, a molecular mass of 697, and the following chemical structure:
A terminal —Cl of the HaloTag-PEG4-biotin ligand is configured to be displaced in a nucleophilic reaction by an Asp106 of the HaloTag protein to form the stable ester bond, whereby forming the biotinylated Halo-Tag.
In an embodiment of the present application, a PEG4 spacer in the HaloTag-PEG4-biotin ligand is further shortened or enlarged to obtain an optimal structural rigidity between the scaffold protein and the target protein.
In an embodiment of the present application, before the step of enabling the target protein to contain the tag, the method further comprises: molecular modeling, during which, a proper position in the target protein suitable for inserting the biotin tag or the biotinylated protein or polypeptide tag or the Strep-tag is found and adjusted, to obtain an optimal structural rigidity between the scaffold protein and the target protein.
In an embodiment of the present application, during the molecular modeling, spacer amino acids are inserted into an amino acid sequence of the target protein, or flexible amino acid sequences which are functionally not relevant are removed from the amino acid sequence of the target protein.
Advantages of the method for determining a protein structure using cryo-electron microscopy according to embodiments of the present application are summarized as follows:
In the method of the present application, a target protein is enabled to contain a tag. A resulting target protein containing the tag is allowed to bind with a scaffold protein to form a complex. Then single-particle imaging is performed using cryo-electron microscopy to determine the structure of the target protein. The tag is capable of selectively binding to the scaffold protein. In this way, the method is generic for structure determination of many kinds of soluble proteins or membrane proteins. Thereby, the target protein may not be restricted to symmetric structures and large molecular mass. In contrast, the method of the present application can realize the structure determination of target proteins of molecular mass down to at least 20 kDa, and the result of the determination is not affected by the structure symmetry of the target protein.
It is another objective of the present application to provide use of HaloTag-PEG4-biotin ligand in the above-mentioned method for determining a protein structure using cryo-electron microscopy. The HaloTag-PEG4-biotin ligand has a molecular formula of C31H57ClN4O9S, a molecular mass of 697, and the following chemical structure:
In an embodiment of the present application, a terminal —Cl of the HaloTag-PEG4-biotin ligand is configured to be displaced in a nucleophilic reaction by an Asp106 of a HaloTag protein to form a stable ester bond, whereby forming a biotinylated Halo-Tag.
Advantages of the HaloTag-PEG4-biotin ligand according to embodiments of the present application are summarized as follows:
The terminal —Cl of the HaloTag-PEG4-biotin ligand can be displaced in a nucleophilic reaction by the Asp106 of the HaloTag protein to form a stable ester bond, so as to form the biotinylated Halo-Tag. The biotinylated Halo-Tag is capable of connecting the target proteins to the scaffold protein to form a protein complex, which is suitable to perform the single particle cryo-EM to determine the structure of the target protein. The length of the PEG4 spacer in the HaloTag-PEG4-biotin ligand might be changed to minimize flexibility between HaloTag protein and streptavidin scaffold protein.
The present application is described hereinbelow with reference to the accompanying drawings, in which:
To further illustrate the present application, experiments detailing a method for determining a protein structure using cryo-electron microscopy are described below. It should be noted that the following examples are intended to describe and not to limit the present application
Where an indefinite or definite article is used when referring to a singular noun, such as “a” or “an”, “the”, unless otherwise specified, include the plural form of the noun. When the term “comprising” is used in this specification and claims, it does not exclude other elements or steps. In addition, the terms first, second, third, etc. in the specification and claims are used to distinguish similar elements, and not necessarily used to describe order or time sequence. It should be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein can operate in other orders than described or illustrated herein. The following terms or definitions are provided only to help understand the present invention. Unless specifically defined herein, all terms used herein have the same meaning as understood by those skilled in the art of the present invention.
As used herein, the term “target protein” or “protein of interest” refers to a protein whose structure is to be determined.
“scaffold protein” used herein refers to proteins having multiple protein binding domains that bind specifically a tag or a modified tag attached to the protein whose structure is to be determined.
The terms “protein”, “polypeptide” and “peptide” are further used interchangeably herein to refer to polymers of amino acid residues and their variants and synthetic analogs.
Biotin is hexahydro-2-oxo-1H-thieno(3,4-d)imidazole-4-pentanoic acid. It is also known as vitamin H and coenzyme R, and is a water-soluble vitamin and also belongs to the vitamin B family, B7. It is a necessary substance for the synthesis of vitamin C and an indispensable substance for the normal metabolism of fat and protein.
Streptavidin is a protein complex made up of four identical protein subunits. The tetramer has a molecular mass of 52 kDa protein and can be purified from the bacterium Streptomyces avidinii or can be produced by heterologous protein expression in E.coli. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin H). With a dissociation constant (Kd) on the order of ≈10−14 mol/L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Streptavidin is used extensively in molecular biology, bioanalysis and biotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants (e.g. guanidinium chloride), detergents (e.g. SDS, Triton X-100), proteolytic enzymes, and extremes of temperature and pH.
Water soluble proteins of interest in the present context having a molecular mass below 50 kDa include enzymes, transcription factors and transport proteins, are found free in cellular compartments such as the cytoplasm, nucleus, or endoplasmic reticulum.
Integral membrane proteins, briefly mentioned herein as “membrane proteins”, are proteins that are embedded among the lipids that make up the bilayer structure of cell membranes. The membrane proteins perform specific tasks that are essential for the proper functioning of the cell. These include translocation of molecules and ions into and out of the cell, detecting extracellular signals and transmitting them into cells. The extracellular sites of membrane proteins are often glycosylated making the cell recognizable to other cells.
G protein-coupled receptors (GPCRs) are integral membrane proteins that transmit physical and chemical signals from the extracellular space into the cell. The chemical signals recognized by GPCRs range from small molecules like neurotransmitters to hormones and proteins. There are about 800 representatives in the human proteome GPCRs establish the largest family of membrane proteins. There are five different classes of GPCRs in humans. The rhodopsin-like class A contains the largest number of GPCRs with about 700 representatives. About half of them are olfactory receptors. The molecular mass of typical class A GPCRs are in the range of 30 to 40 kDa. Each receptor contains a transmembrane domain composed of seven α-helices. The extracellular N-terminus is often glycosylated. Specific conserved glycosylation sites can be selectively biotinylated by a one-step chemical reaction. The intramembrane C-terminus contains often an α-helix which is attached via lipid anchors to the intracellular side of the plasma membrane. GPCRs are among the most important targets for small molecule drugs.
Neurokinin 1 receptor (NK1R) is the main receptor for the tachykinin peptides such as substance P. NK1R belongs to class A GPCRs. It couples to Gq and Gs-protein signaling pathway.
As used herein, the term “fusion”, and interchangeably used herein as “conjugating”, “linking”, especially referring to “genetic fusion”, such as by recombinant DNA technology, and referring to “Chemical and/or enzymatic binding” leading to stable covalent linkage.
As used herein, the term “protein complex” or “complex” or “assembled protein” refers to a group of two or more bound macromolecules, at least one of which is a protein.
The term “binding” means any interaction, whether it is direct or indirect. Direct interaction suggests contact between binding partners. Indirect interaction means any interaction whereby the interaction partner interacts in a complex of more than two molecules.
As used herein, the term “antibody” refers to an immunoglobulin G (IgG) molecule or a molecule containing an immunoglobulin (Ig) domain that specifically binds to an antigen. The antibody may be a whole immunoglobulin from a natural source or from a recombinant source and may be an immunoreactive part of a whole immunoglobulin. IgG antibodies are generally made of four peptide chains.
As used herein, the term “anticalin” refers to proteins which are engineered lipocalins with novel, antibody-like binding functions.
As used herein, the term “label” or “tag” refers to a detectable probe that allows the detection, visualization, and/or separation, purification, and/or immobilization of the (poly)peptide or protein described herein that has been isolated or purified. It is intended to include any marker/label known in the art for these purposes.
The term “wild type” refers to a gene or gene product isolated from a naturally occurring source. The wild-type gene is the most commonly observed gene in the population, so the “normal” or “wild-type” form of the gene is arbitrarily designed. On the contrary, the terms “modified”, “mutant”, “derivative”, or “variant” refer to the sequence, post-translational modification and/or functional characteristics (i.e. changed characteristics) that are displayed when compared with the wild-type gene or gene product or modified gene or gene product.
The terms “vector”, “vector construct”, “expression vector” or “gene transfer vector” as used herein are intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule linked to it, and includes any known to those skilled in the art vectors, including any suitable type, including but not limited to plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as adenovirus, AAV or baculovirus vectors) or artificial chromosome vectors (such as bacterial artificial chromosomes) (BAC), yeast artificial chromosome (YAC) or P1 artificial chromosome (PAC).
Here we present a novel technique to solve the structure of target proteins of molecular mass down to at least 20 kDa. It is generic, i.e. it can be identically used for many different target proteins. The target protein for structure determination comprises a tag which selectively binds to streptavidin, avidin or modified versions thereof (
In general, the tags can be distinguished in two groups summarized in Table 1.
(1) Biotin, directly attached to target protein or biotinylated protein tag or biotinylated polypeptide tag fused to the sequence of the target protein-of-interest.
The fusion protein of interest comprises one of the following tags:
The above-mentioned tags offer the additional advantage to be used for affinity purification of the target protein (https://en.wikipedia.org/wiki/Strep-tag; Lin et al, 2020).
(2) Biotinylated or strep-tagged antibodies or antibody Fab fragments or single-chain antibodies (nanobodies) or anticalins (Gebauer & Skerra, 2020) binding selectively to target protein-of-interest (POI).
In this approach the biotin or the strep-tag sequence is covalently attached to an IgG-type antibody, antibody fragment or nanobody which binds with high affinity and selectively to a native or genetically introduced sequence or structural motive in the POI. This approach is especially of interest if a native, unmodified POI should be assembled to the scaffold protein streptavidin or its derivatives.
There are different approaches to introduce tags to such GPCR/signaling-protein complexes to enable binding of these complexes to the streptavidin or streptavidin-like scaffold protein: (i) using GPCRs comprising tags as described in (
The feasibility of the new approach to use a GPCR assembled via halotag on a streptavidin template for elucidating the structure of the corresponding GPCR by single-particle cryo-EM is demonstrated using the neurokinin 1 receptor (NK1R) as a prototypical GPCR. The high-resolution structures of the human NK1 receptor (NK1R) bound to small-molecule antagonist therapeutics has been solved recently by X-ray crystallography and NMR spectroscopy (Schöppe et al, 2019; Chen et al, 2019). Here, the NK1R serves as a test case for comparing to cryo-EM structures of different streptavidin-(NK1R-halotag)n constructs, with n=1-4, with those of the published NMR and X-ray structures.
Molecular modeling is a very important step in finding a proper position of the biotinylated tag or the strep tag in the protein sequence of the GPCR. In a first step, homology computer modeling will be used to establish a 3D structural model of any GPCR whose structure has to be solved experimentally by the new cryo-EM approach. In a second step, further computer modeling will be used to insert the biotinylated tag of interest on one of the positions of the GPCRs outlined in
It is obvious that the method described before can be easily adapted to solve the structure of water-soluble proteins as targets.
All models were built with the 3D builder tool in Schrodinger Maestro software. The ionization states of protein were assigned properly according to the results from Schrodinger. MD simulations were carried out using the Desmond software. The optimized potentials for the liquid simulations (OPLS)-3e force field were used in this system, the protein was solvated with the simple point charged (TIP3P) water model and 0.15 M NaCl. The orthorhombic water box was used to create a 15 Åbuffer region between the protein atoms and box sides. The temperature was maintained constant at 310 K, and a 2.0 fs value was obtained in the integration step. We investigated the structure by performing 50 ns unbiased molecular dynamics (MD) analysis, and ANM calculation was utilized to analyze the protein backbone motion in MD trajectory.
A HaloTag-PEG4-biotin ligand was synthesized. The HaloTag-PEG4-biotin ligand has a molecular formula of C31H57ClN4O9S, a molecular mass of 697, and the following chemical structure:
A terminal —Cl of the HaloTag-PEG4-biotin ligand is configured to be displaced in a nucleophilic reaction by an Asp106 of the HaloTag protein to form the stable ester bond (Los et al, 2008), whereby forming the biotinylated Halo-Tag.
2-(2-((6-Chlorohexyl)oxy)ethoxy)ethanamine hydrochloride and NHS-PEO4-Biotin were used to synthesis the PEG-Biotin Ligands. To a stirring solution of NHS-PEO4-Biotin (5 mg, 8.5×10−6 mol) in 115 μl dry DMF was added via syringe a 0.3 M solution of 2-(2-((6-Chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85 μl, 2.55×10−5 mol) in CH2Cl2 followed by one drop of diisoproplyethylamine (excess). The reaction mixture was stirred for 4 hours, then diluted to 1 ml of water and subjected to preparative HPLC purification. H2O and acetonitrile were used as solvent for preparative HPLC and optical absorbance was detected at 210 nm. Purified sample was analysed on LC-MS using H2O-formic acid (1%) and acetonitrile as solvent.
A main chemical reaction formula is as follows:
The DNA fragments of wild type halotag protein were amplified by PCR and cloned into a pET29a vector. For protein production, the plasmids were transformed into Escherichia coli BL21 (DE3) cells. The cells were grown at 37° C. until an OD600=0.6 was reached, then the sample was shifted to 18° C., and proteins were induced by adding 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells were harvested using centrifugation after 18 hours post induction and resuspended in a lysis buffer comprising 25 mM Tris-HCl pH 7.5, 400 mM NaCl and 10 mM imidazole. Cells were lysed by passing the cell suspension through a French press (800 p.s.i.). The lysed cell preparation was centrifuged for 20 min at 12,000×g. The pellet was discarded and the supernatant was applied onto nickel affinity column equilibrated with the binding buffer consisting of 25 mM Tris-HCl, pH 7.5, 400 mM NaCl and 10 mM imidazole. The proteins bound on the column resin were first washed with 50 mM imidazole and then eluted with 250 mM imidazole. The obtained protein preparation was dialyzed twice against 25 mM Tris-HCl (pH 8.0), 100 mM NaCl. Finally, size exclusion chromatography was used for isolating the monomeric form of the halotag protein.
The streptavidin (purchased from the IBA, Gottingen, Germany) was mixed with the halotag-PEG4-biotin ligand at the molar ration of 1:4. After 3 hours incubation, monomer of halotag protein was added to the mixture. After overnight incubation, the mixture of streptavidin, halotag-PEG4-biotin ligand and halotag protein was applied to size exclusion chromatography to separate the formed streptavidin-halotag protein complex from the reaction mixture.
Electron microscopy of negative stained samples were used to evaluate the protein quality. In brief, 3 μL of freshly purified streptavidin-halotag protein complex (0.025 mg/ml) was applied onto copper grids supported by a thin layer of glow-discharged carbon film (Zhongjingkeyi Technology Co., Ltd). After adsorption for 1 min, uranyl acetate (2% w/v) was used for negative staining at room temperature. The negative stained grid was examined using FEI Talos L120C operated at 120 kV.
Peak fractions collected from the size exclusion chromatography were concentrated to 1 mg/mL and then centrifuged at 12,000×g for 30 min at 4° C. A total of 4 μL of sample was applied to glow-discharged Quantifoil porous carbon grids (gold, 2-2 μm/well, 300 mesh), blotted using a Vitrobot (FEI) with a blotting time of 2 seconds at a temperature of 4° C. at 100% humidity, and then frozen in liquid nitrogen cooled liquid ethane. Frozen grids were carefully transferred and stored in liquid nitrogen until cryo-EM images were collected.
A total of 19060 movies were collected on a Titan Krios G3i (Thermofisher Scientific) operated at 300 kV equipped with a Gatan image filter Continuum 1069 (operated with a slit width of 20 eV), mounted with a K3 Summit detector (Gatan, Inc.). EPU was used to automatically acquire micrographs in super-resolution counting mode at a pixel size of 0.4275 Å and with nominal defocus values ranging from −1.5 to −2.5 μm. Movies with 32 frames each were collected at a dose of 50 electrons per pixel per second over an exposure time of 2.1 s, resulting in a total dose of 50 e−/Å2 on the specimen. Tilting data at 10°, 20°, 30° and 40° were collected to overcome the preferred orientation issue of the streptavidin-halotag complex. All movie frames in each stack were aligned and dose weighted using MotionCor2, which generated 2-fold binned images to a pixel size of 0.855 Å/pixel. The patch ctf module of Cryosparc2 software was used for estimating the defocus values and astigmatism parameters of the contrast transfer function (CTF). A total of 12655 micrographs were chosen for further processing. To investigate the subunit organization, reference-free two-dimensional (2D) classifications were performed. The 2D classification module of Cryosparc2 was used for further data processing. 5850507 particles were initially picked from selected micrographs and subjected to 2D classification. After performing 2D classification in Cryosparc, the best-looking 2D class averages, as judged by visual inspection, were selected to build an ab initio reconstruction for heterogeneous refinement in cryoSPARC. After heterogeneous refinement, one class showing intact features (particles) was selected and subjected to nonuniform refinement with C2 symmetry, followed by local refinement, yielding an overall resolution of 3.6 Å.
The DNA coding sequence of the Halotag protein was inserted into the intracellular loop 3 of the coding sequence of the NK1R and then the construct was amplified by PCR and cloned into a pEG BacMam vector. HEK293F cells were cultured in SMM 293T-II medium under 8% CO2 in an incubation shaker at 37° C. Target protein was expressed by transient transfection of the HEK293F cells using plasmid DNA coding for the NK1R-halotag fusion protein. Briefly, for 1 liter culture of HEK293F cells, 1 mg plasmid DNA and 4 mg 25-kDa linear polyethylenimines were preincubated for 10 min in 25 ml fresh medium, and then two media samples were mixed for further incubation for 15 min prior to adding the mixture to cells. The transfected cells were cultured for 72 h before harvest by centrifugation. For each batch of protein purification, 4 liters of transfected HEK293F cells were harvested by centrifugation at 2000×g. Cell pellets were resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 500 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.1 mM PMSF and 1×protease inhibitor cocktail and disrupted by French press. Cell membranes were solubilized in lysis buffer containing 1% (wt/vol) N-dodecyl-β-D-maltoside (DDM), 0.2% Cholesteryl Hemisuccinate Tris Salt (CHS) and 0.5 μM netupitant for 12 h with stirring at 4° C. Solubilized NK1R-halotag protein was separated from the insoluble fraction by centrifugation for 1 h at 40,000×g and incubated with 2 ml of TALON metal affinity resin for 3 h at 4° C. The resin was then washed with 10 column volumes of buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 0.5 μM netupitant, 0.1 mM PMSF and 0.1% DDM, 0.02% CHS and 30 mM imidazole. Resin was eluted by 5 column volumes of buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 0.5 μM netupitant, 0.1 mM PMSF and 0.1% DDM, 0.02% CHS and 250 mM imidazole. The eluted protein was collected and concentrated using a 100 kDa concentrator. The streptavidin (purchased from IBA, Gottingen, Germany) was mixed with the Halotag-PEG4-biotin ligand at the molar ration of 1:4 to bind four Halotag-PEG4-biotin ligands to one streptavidin tetramer, named SA(Halotag PEG-Biotin ligand)n (n=1-4). After 3 hours incubation, NK1R-Halotag fusion protein was added to the mixture to covalently bind to the halotag ligand of SA(Halotag PEG-Biotin ligand)n. After overnight incubation, the reaction mixture was applied to size exclusion chromatography to separate the formed streptavidin-NK1R-halotag complex from the reaction mixture.
The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. While particular embodiments of the invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the invention in its broader aspects, and therefore, the aim in the appended claims is to cover all such changes and modifications as fall within the true spirit and scope of the invention.
This application claims priority to pending U.S. Provisional Application No. 63/335,954 filed on Apr. 28, 2022, the contents of which are incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63335954 | Apr 2022 | US |
