Patent Application
20060134604
1. Field of the Invention
The present invention relates to flexible high-throughput methods and apparatus for expressing, extracting and purifying relatively large quantities of predetermined recombinant proteins. The invention further relates to a method and apparatus for purifying a plurality of predetermined proteins simultaneously in separate but parallel operating apparatuses. The invention further relates to a method and apparatus for tracking, planning and maintaining a production system for producing a plurality of predetermined proteins simultaneously. The invention further relates to production and purification of a plurality of proteins for use in personalized medicine. The invention further relates to flexible production and purification of a plurality of proteins for use in microarrays. The invention further relates to production and purification of a plurality of proteins for use in protein related research.
2. Background of the Related Art
The study and use of proteins has gained prominence in both the scientific and medical communities in the last few decades as both physicians and researchers recognize the important role proteins play in the physiological and metabolic functions within organisms, such as human beings. Many aspects of proteins are continually being studied, such as protein-protein interactions, glycosylations, identification of protein disease related markers and other characteristics. Proteins are being used in microarrays for use in both research and clinical applications, and large quantities of proteins are required for the production and characterization of antibodies. Hence, the production of proteins is becoming critical for further development in these areas.
There are many protein production techniques, each having its own advantages and limitations. Such methods for producing full-length or partial length proteins include: bacterial based systems, yeast based systems, fungi based systems, insect based systems, mammalian systems and plant systems, such as the GENEWARE® system developed by Large Scale Biology Corporation in Vacaville, Calif.
For all protein systems expressing heterologous proteins, cDNA or DNA sequences of interest are first cloned into a suitable vector which is capable of being transcribed or induced in the host species transformed with the vector DNA. For example, bacterial based systems utilize plasmid, phage or viral-derived vectors for expression of heterologous proteins. Vector DNA containing the nucleic acid sequence of interest (insert DNA) is inserted into the bacteria through standard transformation techniques, including calcium phosphate and electroporation transformation. In addition, many kits are available for the insertion of isolated and purified insert DNA into the selected vector system, making bacterial systems the most widely used for routine expression and purification of heterologous proteins. Although bacterial based systems are frequently used to express heterologous proteins in relatively large quantities, problems of proper folding and lack of post-translational processing may produce functionally inactive molecules. Traditionally, bacterial based systems, therefore, are suitable for only a small range of proteins.
Insect based, and to a lesser extent yeast-based, systems may permit folding, post-translational modification and oligomerization similar to that seen of the native heterologous protein, but fall short of the complexity exhibited by native proteins. An example of an insect based system for producing proteins is the use of baculovirus in insect cells. Plasmid-based Drosophila cell systems are also available, which obviate the necessity for the manipulation and maintenance of baculovirus. Both baculoviral and plasmid-based Drosophila systems utilize vectors, similar to bacterial based systems, for insertion and subsequent expression of heterologous proteins in the host cell. Yeast systems also utilize DNA vectors, such as commercially available pESC, pYES, pNMT, pYD, pPIC and pGAP.
Mammalian expression systems, such as mammalian cell cultures (e.g. NIH 3T3, HeLa, K562, 293 and other cell cultures) transfected with plasmid or phage-based vectors or infected with viral vectors, are capable of substantial post-translational modification. Examples of commercially-available vectors used in mammalian expression systems include viruses, such as adeno associated virus, pFB retroviral vectors and adenovirus, plasmids, such as pACT, pBIND, pCAT, pCI, phRG-CMV, phRG-TK, phRL-TK, pSI and pERV, and phage-based vectors, including pBK, pBK-CMV and pBK-RSV. Mammalian cells, however, may be more problematic to expand to larger scale capabilities because of the culture-intensive work required for expressing foreign proteins. In addition, technical expertise may be required for producing enough cells with the desired quantity of protein. For example, mammalian cells, in particular, may require stable transformation and chromosomal integration of vector DNA because of the inefficiency of transient transfections.
Proteins expressed in plant-based systems also require vectors for the expression of heterologous proteins. For example, Donson et al, U.S. Pat. No. 5,316,931 and U.S. Pat. No. 5,589,367, herein incorporated by reference, demonstrate plant viral vectors suitable for the systemic expression of foreign genetic material in plants. Donson et al. describe plant viral vectors having heterologous subgenomic promoters for the systemic expression of foreign genes. The availability of such recombinant plant viral vectors makes it feasible to produce proteins and peptides of interest recombinantly in plant hosts.
Isolation of proteins produced in bacteria, yeast, insect (baculovirus) and mammalian cultures is also well known. For instance, Qiagen, Valencia Calif., markets materials such as metal affinity resins and magnetic beads compatible with 96-well plate formats for the purification of 6×His-tagged proteins. Such purification techniques are described in A Handbook For High Level Expression And Purification Of 6×His-tagged Proteins published by Qiagen March 2001, and further disclosed in the US Patent Numbers: U.S. Pat. Nos. 4,877,830, 5,047,513, 5,284,933 and 5,310,663, all of which are incorporated herein by reference. However, many of the methods disclosed in the above group of patents and the materials sold by Qiagen, are optimized for isolating quantities of proteins measured in μg or less (not mg quantities) and are further not specifically designed for purification of proteins produced in plants.
Some processes for isolating proteins, peptides and viruses from plants have been described in the literature (Johal, U.S. Pat. No. 4,400,471, Johal, U.S. Pat. No. 4,334,024, Wildman et al., U.S. Pat. No. 4,268,632, Wildman et al., U.S. Pat. No. 4,289,147, Wildman et al., U.S. Pat. No. 4,347,324, Hollo et al., U.S. Pat. No. 3,637,396, Koch, U.S. Pat. No. 4,233,210, and Koch, U.S. Pat. No. 4,250,197, the disclosures of which are herein incorporated by reference in their entirety).
Methodologies have been developed for the cost-effective and large-scale purification of bioactive species produced in plants. These bioactive species may be proteins or peptides, especially recombinant proteins or peptides, or virus particles, especially genetically engineered viruses. Specifically, U.S. Pat. No. 6,037,456 to Garger et al., discloses methods for isolation and purification of large quantities of a protein extracted from, for instance, tobacco plants that have been infected with a recombinant tobacco mosaic virus. The methods disclosed in U.S. Pat. No. 6,037,456 are generally intended for isolation and purification of proteins from large quantities of tobacco plant or other acceptable plant material, where the quantity of protein isolated may be measured in hundreds of grams to kilograms. Further, co-pending and commonly assigned patent application “Flexible Processing Apparatus for Isolating and Purifying Viruses, Soluble Proteins and Peptides from Plant Sources” application Ser. No. 09/970,150 filed Oct. 3, 2001, discloses an automated apparatus for purification of large quantities of proteins produced in plants, again where the quantity of proteins isolated are measurable in hundreds of grams to kilograms. Although the methods described in the patent and pending patent application have many advantages, they are meant for large scale production of material and are not easily applicable to isolation and purification of smaller, more modestly sized quantities of a plurality of proteins, where the quantity of each individual protein is measured in micro-grams to milligrams. U.S. Pat. No. 6,037,456 and co-pending and commonly assigned patent application “Flexible Processing Apparatus for Isolating and Purifying Viruses, Soluble Proteins and Peptides from Plant Sources” application Ser. No. 09/970,150 filed Oct. 3, 2001, are both incorporated herein by reference in their entirely.
There is a need for a flexible system for production and purification of multiple proteins where the proteins may be produced in any of a variety of cultures, and where the proteins are purified in a reliable manner and provide desired quantities of each protein. There is also a need for methods and apparatuses that efficiently perform the production and isolation of 100's μg to several mg of recombinant protein from plant material where the starting biomass ranges from 10 g to less than 10 kg. There is also a need for methods and apparatuses that efficiently perform the production and isolation of similar quantities of recombinant protein produced by bacteria, insect, mammalian and/or yeast cultures. There is also a need for methods and apparatuses that may efficiently perform the production and isolation of proteins associated with proteins of interest to determine proteome structure and relationships within a defined cell, tissue or host organism.
Further, advances in human genome research are opening the door to a new paradigm for practicing medicine that promises to transform healthcare. Personalized medicine, the use of marker-assisted diagnosis and targeted therapies derived from an individual's molecular profile, may impact the way drugs are developed and medicine is practiced. The traditional linear process of drug discovery and development may soon be replaced by an integrated and heuristic approach. Current practice among pharmaceutical manufacturers is to produce massive amounts of a single pharmaceutical, with statistical evidence demonstrating that the pharmaceutical product of interest will only be able to treat a portion of the patient population due to undesirable and adverse reactions in the remaining portions of the target patients. There is a need for production of pharmaceutical products on a small scale where medicines are produced that are tailored to a specific individual or patient population.
Where the virus or protein isolated is intended for production as a pharmaceutical product, consistent and verifiable methodology is required. Therefore, there is a need for automated methodology and apparatus for isolating proteins where the automated apparatus monitors and provides tracking and verification of methodology used in the isolation process.
The invention relates to a multiple channel apparatus for parallel and simultaneous purification of a plurality of separate proteins.
The present invention also relates to method and apparatus for simultaneous production and purification of a plurality of proteins.
Definitions
In order to provide a clear and consistent understanding of the specification and the claims, including the scope given herein to such terms, the following definitions are provided:
GENEWARE® is a technology developed by Large Scale Biology Corporation, located in Vacaville Calif., to test the function of novel genes and proteins they encode, and to manufacture complex proteins in bulk. GENEWARE® includes the use of a vector modified from a virus to place any gene or a large number of genes within a test organism. The organism then manufactures the gene's protein product, which can be studied, collected and purified.
Preferably, GENEWARE® utilizes tobacco plants or related Nicotiana species infected with a transgenic tobacco mosaic virus. GENEWARE® technology typically includes the use of tobacco plants because the quick-growing tobacco plant provides an extremely useful model organism for studying plant genes, as well as a high-yield factory for manufacturing any protein, either animal or plant, in bulk. A variety of aspects of the GENEWARE® technology are disclosed in the following US Patents commonly assigned to Large Scale Biology Corporation, which are incorporated herein by reference in their entirety: U.S. Pat. No. 5,316,931 to Donson et al., U.S. Pat. No. 5,589,367 to Donson et al., U.S. Pat. No. 5,766,885 to Carrington et al., U.S. Pat. No. 5,811,653 to Turpen, U.S. Pat. No. 5,866,785 to Donson et al., U.S. Pat. No. 5,889,190 to Donson et al., U.S. Pat. No. 5,889,191 to Turpen, U.S. Pat. No. 5,922,602 to Kumagai et al., U.S. Pat. No. 5,965,794 to Turpen, and U.S. Pat. No. 6,054,566 to Donson et al. However, it should be understood that the GENEWARE® technology is applicable to use with plants other than tobacco, such as corn, rice, etc.
In the following description, the terms “bio-mass”, “bio-matter” and “plant source” all refer to any harvested plant, seed or portion of a plant that may be processed to extract or isolate material of interest such as viruses, proteins and/or peptides therefrom. For instance, the bio-matter process may include many types of plants or portions of plants such as seeds, flowers, stalks, stems, roots, tuber, as well as leaf portions of plant material. Typically, the succulent leaves of tobacco plants are ideal for large scale production of predetermined proteins using GENEWARE® technology, but it should be understood from the following description that plants other than tobacco may be used for the production of proteins using GENEWARE® technology.
Alternatively, other plants such as corn, rice, grains or other desirable plants may be utilized for the production of proteins and peptides of interest.
In the following description, the terms “bio-mass” and “bio-matter” may also refer to biological material produced by bacterial based systems, insect based systems, mammalian systems and yeast systems, where the biological material is harvested for the purpose of purifying proteins produced therein in accordance with the methodologies set forth in the description below.
The term “green juice” refers to liquid extracted from processed bio-matter. However, it should be understood that the term green juice may refer to any liquid extracted from a plant material or bio-matter regardless of the extracted liquid's color. For instance, where a protein or proteins is produced using Large Scale Biology Corporation's GENEWARE® technology, the green juice may indeed be green where the green juice originated from bio-matter such as harvested tobacco. However, where proteins of interest are expressed by bacterial based systems, insect based systems, mammalian systems, fungi systems and yeast systems, the liquid extracted therefrom may not have a green color, but in the description below may still be referred to as green juice.
A “virus” is defined herein to include the group consisting of: a virion wherein the virion includes an infectious nucleic acid sequence in combination with one or more viral structural proteins; a non-infectious virion wherein the non-infectious virion includes a non-infectious nucleic acid in combination with one or more viral structural proteins; and aggregates of viral structural proteins wherein there is no nucleic acid sequence present or in combination with the aggregate and wherein the aggregate may include virus-like particles (VLPs). The viruses may be either naturally occurring or derived from recombinant nucleic acid techniques and include any viral-derived nucleic acids that can be adopted whether by design or selection, for replication in whole plants, plant tissues or plant cells.
A “virus population” is defined herein to include one or more viruses as defined above wherein the virus population consists of a homogenous selection of viruses or wherein the virus population consists of a heterogenous selection including any combination and proportion of the viruses.
“Virus-like particles” (VLPs) are defined herein as self-assembling structural proteins wherein the structural proteins are encoded by one or more nucleic acid sequences wherein the nucleic acid sequence(s) is inserted into the genome of a host viral vector.
“Protein and peptides” are defined as being either naturally-occurring proteins and peptides or recombinant proteins and peptides produced via transfection or transgenic transformation.
The terms “protein of interest” “material of interest” and “materials of interest” refer to any material, compound, organic structure or combination of materials to be isolated using the purification methods and/or apparatus in accordance with the present invention. The protein, material or materials of interest may include, but are not limited to: virons, virus-like particles, viruses, proteins and/or peptides, receptors, receptor antagonists, antibodies, single-chain antibodies, enzymes, neuropolypeptides, insulin, antigens, vaccines, peptide hormones, calcitonin, and human growth hormone. Further, the protein, material or materials of interest may be an antimicrobial peptide or protein consisting of protegrins, magainins, cecropins, melittins, indolicidins, defensins, 13defensins, cryptdins, clavainins, plant defensins, nicin and bactenecins.
A “bacteria” is defined herein to include the group consisting of small, unicellular microorganisms that multiply by cell division and whose cell is typically contained within a cell wall, occurring in spherical, rodlike, spiral, or curving shapes and found in virtually all environments.
A “bacterial culture” is herein defined as the maintenance and reproduction of a bacterial population in vitro. The bacterial population is typically clonal in origin, i.e. derives from a single bacterial cell. Therefore, all bacteria within a given bacterial culture should contain the same genetic complement, and in the case of protein expression systems, express the same heterologous protein sequence. The bacterial culture, however, may, in certain circumstances, originate from more than one bacterial cell, and therefore contain a plurality of bacterial cells with differing genetic complements.
A “mammalian cell” is herein defined to include the group consisting of cells derived from a mammalian origin. Sources of mammalian cells include, but are not limited to, tissue, fluids, blood, organs or other biological sources from humans and other mammals.
A “mammalian cell culture” is herein defined to include the group of cells derived from a mammalian source capable of surviving ex-vivo in a cell culture medium. The mammalian cell may be a primary cell, directly derived from a mammalian cell source. More typically, the mammalian cell in a mammalian cell culture will be immortalized, i.e. capable of growth and division through an indeterminate number of passages or divisions.
A “yeast cell” is herein defined to include the group consisting of small, unicellular organisms capable of growth and reproduction through budding or direct division (fission), or by growth as simple irregular filaments (mycelium). The yeast cell may be transformed or transfected with a heterologous vector for expression of a nucleic acid sequence inserted into the heterologous vector. An example of a yeast cell includes Saccharomyces cerevisiae, commonly used for transfection and expression of heterologous proteins.
An “insect cell” is herein defined to include the group of cells derived from an insect source capable of surviving ex-vivo from an insect host. The insect cell may be transformed, transfected or infected with a heterologous vector for expressions of a protein sequence inserted into the heterologous vector. Examples of insect cells include High Five™ cells, Aedes albopictus cells, Drosophila melanogaster cells and Mamestra brassicae cells.
An “affinity tag” is a molecule, ligand or polypeptide attached to a protein (polypeptide) of interest. Examples of affinity tags include, but are not limited to, hexahistidine, other metal tags, streptavidin, biotin, specific epitope markers for antibody purification, glutathione-S-transferase, β-galactosidase, β-amylase and other protein or small molecule tags which may assist in the isolation and purification of expressed proteins.
An “affinity matrix” is a solid-state material bound to a substrate or ligand, which in turn binds selectively to an affinity tag attached to a protein of interest. Upon binding of the affinity tag to the affinity matrix, the protein of interest is retained within the column or other purifying apparatus, and may thus be separated from any impurities present in the green juice. After washing of the affinity matrix, the protein of interest, with the affinity tag attached, may be eluted from the column or other apparatus in a substantially purified form. Examples of affinity matrices include chromatography medium, such as agarose, cellulose, Sepharose, Sephadex and other chromatography medium, polystyrene beads, magnetic beads, filters, membranes and other solid-state materials bound to ligands or substrates which bind to the affinity tag of choice.
A “histidine-tagged protein” is a protein of interest whereby a histidine affinity tag is attached either at the carboxy-terminus, amino terminus or internal to the protein of interest. Typically, the histidine tag consists of six histidine moieties, but may consist of any combination or numerical designation of histidine moieties. The histidine-tagged protein is purified by binding the histidine-tagged protein to a metal affinity matrix, such as Ni-NTA Agarose (manufactured by QIAGEN, Inc.), and washing impurities from the bound affinity matrix. The histidine-tagged protein can then be eluted from the column using acid pH buffering conditions, competitive elution by imidazole or by stripping the metal from the affinity matrix using EDTA (ethylene diamine tetra-acetate).
Overview (
In accordance with the present invention, a protein or proteins of interest are produced by any of a variety of methods, as indicated in
In a first step, shown at box S1 in
In the description below, production and purification of at least one protein is described in detail. For most of the following description, production and purification of only one protein is included in order to simplify the description, eliminate redundant language and make this description easier to follow. However, it should be understood from the following description that a plurality of proteins are produced and purified simultaneously in accordance with the present invention.
At box S2 in
At box S3 in
A determination is made at box S4 in
As represented at box S5 in
After purification, the purified protein of interest is tested to confirm characteristics and consistency, as represented at box S8 in
Protein & Insert Selection (
There are a variety of processes through which protein or proteins of interest may be selected for production and purification, dependent upon the function or purpose thereof. The protein or proteins of interest may be patient specific medicines such as vaccines as described in co-pending U.S. patent application Ser. No. 09/522,900, filed Mar. 10, 2000, where a patient's own DNA provides a sequence for expression of a specific protein. The proteins of interest may alternatively be target proteins for use in, for instance, microarrays or so called protein chips. Protein targets may be chosen to allow evaluation of physiological parameters from collected specimens (blood, serum, urine, sputum, cerebrospinal fluid or any other biological sample), organ function or dysfunction as well as identification of various pathological infectious states.
Where a sequence is needed to express a specific or known protein (for instance, a sequence that is not specifically taken from a patient), the required sequence may be isolated from various databases, both public and proprietary, using a computer system such as that depicted schematically in
Gene sourcing, or the isolation of nucleic acids of interest, may be produced by a variety of methods, including polymerase chain reaction (PCR), reverse-transcriptase polymerase chain reaction (RT-PCR), colony screening and nucleic acid synthesis. The databases and literature mentioned above, in addition to allowing a researcher or clinician to select proteins expressed for a given state, also contain nucleotide and protein sequence information, allowing suitable target probes to be designed to isolate target cDNA's and proteins of interest.
Considerations of probe design and reaction conditions for isolation are important for isolating specific proteins of interest, known or unknown. Probes to isolate cDNA's of interest may be designed according to protein or DNA sequence information provided by the databases and literature mentioned above. Alternatively, tryptic peptide information from previously unknown proteins isolated on 2-D gels or other methods of protein fractionation and isolation may also be used in probe design. The probes may be synthesized using standard phosphoramidite chemistry, or other nucleic acid synthesis chemistry, incorporating standard deoxynucleotide compounds (dATP, dGTP, dCTP, dGTP), or alternatively may use modified nucleotides that are capable of hybridizing with two or more different deoxynucleotides (dITP or other modified nucleotides). If protein sequences are used as templates for nucleic acid probes, probe sets may consist of at least one pair of primers coding for one permutation of nucleic acid sequence. Alternatively, due to the degeneracy of the amino acid code, more than one pair of primers coding for alternative permutations of the corresponding nucleic acid sequence may be employed. For example, lysine is encoded by two different codon sequences: AAA and AAG. Therefore, a sequence incorporating the amino acid lysine would include both variations within a probe at the lysine position. In addition to synthesizing probes, nucleic acid fragments excised from larger nucleic acid sequences (e.g. cloning vector fragments and other nucleic acid fragments) may also be employed as probes in target nucleic acid isolation.
Probes may also be designed that are similar, but not identical to, known protein sequences. These probes may isolate related proteins that may differ in amino acid sequence composition between individuals, and therefore isolation of such proteins may be difficult using standard probe design techniques. Alternatively, DNA may be screened with nucleic acid probes using decreased stringency conditions, which would allow for the isolation and purification of related, but not identical, DNA sequences. The nucleic acid probes may be used in RT-PCR isolation and cloning from mRNA or total RNA samples. The nucleic acid probes may also be used in genomic DNA cloning from total genomic DNA using PCR amplification or other isolation methodology. Total RNA or genomic DNA may be isolated from animal, plant or bacterial/microbial cells or tissue using standard RNA or DNA purification techniques, e.g. detergent or alkaline lysis, guanidium isothiocyanate, CsCl gradients, Phenol/SDS, Phenol/Chloroform, glass- or silica-based chromatography or other methods, including readily available commercial kits from a variety of manufacturers. Total RNA may be further fractionated on oligo-dT columns or resins to yield poly-A containing mRNA. In addition, mRNA may be directly isolated from cell culture or tissue lysates using standard lysis protocols (alkaline lysis, detergent lysis, mechanical disruption and other lysis methodologies) combined with oligo dT column chromatography.
cDNA strands from reverse transcription of RNA may be copied using DNA polymerase or other available polymerases to yield double-stranded DNA. A variety of standard molecular biology techniques using DNA polymerases may then be used to amplify the double stranded DNA, insert and ligate the amplified cDNA into the appropriate expression vector for further analysis. Alternatively, genomic DNA may be directly PCR amplified using DNA polymerase or other available polymerases. As with amplified cDNA, amplified genomic DNA can be inserted and ligated into the appropriate expression or replication vector for further analysis.
An alternative protocol for isolating a DNA sequence of interest is the synthesis of an insert sequence, and its complementary binding strand, through standard DNA synthesis protocols. For example, complementary DNA strands may be synthesized using standard phosphoramidite chemistry. For cloning purposes, assymetric restriction enzyme sequences may also be incorporated into the synthesized strand for directional cloning into a replication and/or expression vector. Alternatively, blunt end ligation of restriction enzyme linkers after annealing of the DNA strands may be accomplished using standard molecular biology ligation protocols. Using DNA synthesis methodologies, picogram, nanogram, microgram or milligram quantities, usually dependent upon the length of the sequence, may be synthesized and purified, which may avoid potential amplification artifacts that may be introduced with DNA polymerase enzymes. DNA synthesis may also be combined with PCR amplification to amplify sufficient quantities for DNA insertion and subsequent replication of DNA into the appropriate vector.
Yet another method is the use of colony screening of bacterial hosts containing a plurality of vector inserts. Typically, the vector inserts may comprise a plurality of nucleic acid sequences isolated from a specific host tissue, organ or condition. For example, commercial bacterial “libraries” are available that correspond to a plurality of vector inserts from mouse liver, or mice that are phenotypic for a specific disease. Isolated probes from above may be used to screen a large number of bacterial clones transferred onto a solid medium, such as nitrocellulose or nylon filters or membranes. The bacterial clones on the solid medium are lysed, and the DNA contained within each clone denatured and bound to the medium, so that the pattern of colonies is replaced by an identical pattern of bound DNA. The medium is then hybridized to labeled probes which identify the clone containing the DNA sequence of interest. The clone is isolated, amplified by large scale culture and the DNA isolated and excised for manipulation into other vectors of interest.
Vector Selection
In accordance with the present invention, the isolated DNA sequence of interest is inserted into a vector to allow the production of recombinant proteins of interest by any of a variety of methods, such as bacterial based systems, insect based systems, mammalian systems and yeast systems or by using aspects of GENEWARE® technology, as described above and in the above identified patents commonly assigned with the assignee of the present invention. Specifically, in the GENEWARE® system, a virus is genetically manipulated to include a vector, a tag and the genetic sequence or insert of interest, selected specifically for the protein it encodes. The virus is then applied to leafy plant tissue such as the leaves of a tobacco plant, thereby infecting the organism. The plant and virus work to express the specific protein, and the protein is subsequently extracted from the plant tissue and then purified. This basic workflow of the methodology of the present invention is described in greater detail below along with a detailed description of apparatus used to effect the methodology of the present invention. Further, a computer system is also described for tracking the work flow and assisting in determining various aspects of the process in a manner described more clearly below.
In accordance with the present invention, where the GENEWARE® technology is employed, specific vectors and inserts are selected for insertion into a tobacco mosaic virus or other suitable virus. One insert is selected for the specific protein encoded by the genetic sequence of that insert, as is indicated at S1 in
As mentioned above, a variety of cloning and expression vectors may be employed for use in protein expression and purification, depending upon the host system used. Typically, cloning and expression vectors are only able to transfect, transform or infect one specific host system (e.g. only plants or bacteria). However, there are cloning and expression vectors, by the nature of the nucleic acid sequences contained within, which are capable of transfecting, transforming or infecting a plurality of host systems. Those of ordinary skill in the art will appreciate that vectors may be designed to transfect, transform or infect a variety of host systems, and any vector capable of transfecting, transforming or infecting and subsequently expressing the vector insert nucleic acid sequence within the host is contemplated within the scope of this invention.
As mentioned above, the choice of vector used is dependent upon the host system contemplated in the purification procedure. For example, plant systems may use viral vectors, derived either from RNA or DNA viruses, for the introduction and expression of heterologous protein sequences. RNA viral vectors are preferred for their high expression levels and host ranges. U.S. Pat. No. 5,316,931, which is incorporated in its entirety herein by reference, describes plant viral vectors having heterologous subgenomic promoters which allow systemic infection of plant hosts and stable transcription or expression in the plant host of foreign gene sequences. Similarly, U.S. Pat. No. 5,811,653, which is incorporated herein by reference, describes an RNA viral vector from the tobamovirus group capable of overexpressing genes in tobacco plants. U.S. Pat. No. 5,977,438, which is also incorporated herein by reference, describes an RNA viral vector which fuses foreign genes to RNA viral proteins (e.g. coat protein), producing relatively large amounts of foreign protein in the form of a fusion protein.
A preferred embodiment may be an RNA viral vector from the tobamovirus family. An example of this is found in the tobacco mosaic virus-derived GENEWARE vector. In the GENEWARE vector, the TMV Replicase coding sequence is upstream of the coding sequence for TMV movement protein. A cDNA ORF (open reading frame), which is ligated 3′ of the TMV movement protein, is joined in frame to a hexahistidine affinity tag polypeptide coding sequence or any other affinity tag coding sequence either at the 3′ or 5′ end. The addition of an affinity tag coding sequence within the cloning and expression vector allows the purification of proteins from complex mixtures by binding the affinity tag-protein of interest to an affinity matrix and subsequently washing the same until all impurities are removed. The protein and affinity tag can then be eluted from the affinity matrix in a substantially pure form. The vector may be optimized for higher expression in protoplasts and inoculated leaves, may be cloning friendly with multiple restriction enzyme sites in the polylinker region 5′ of the cDNA insertion site and contain termination sequences for proper termination of the expressed protein. For example, a tobacco mosaic virus-derived vector may include the TMV replicase coding sequence, which may substantially increase expression in both protoplasts and inoculated leaves. In addition, restriction enzyme sites, including EcoRI, BamHI, SmaI, SacI, NotI, XbaI, SpeI, XhoI, Sap I or other restriction enzyme sites may be contained within a multiple cloning site polylinker sequence flanking the insertion site of the desired nucleic acid sequence. Other RNA viral vectors besides tobamovirus vectors may also be employed, including, but not limited to, rice dwarf virus, wound tumor virus, turnip yellow mosaic virus (tymovirus), rice necrosis virus, cucumber mosaic virus (cucumovirus), barley yellow dwarf virus (luterovirus), tobacco ringspot virus (nepovirus), potato virus X (potexvirus), potato virus Y (potyvirus), tobacco necrosis virus, tobacco rattle virus (tobravirus), tomato busy stunt virus (tombusvirus), watermelon mosaic virus, brome mosaic virus (bromovirus) and other RNA viruses. The RNA in single-stranded RNA viruses may be either a plus (+) or a minus (−) strand.
DNA viral vectors may also be employed for subsequent inoculation and protein expression in host plants. Examples of DNA viral vectors include, but is not limited to, caulimoviruses such as Cauliflower mosaic virus, Cassaya latent virus, bean golden mosaic virus, Chloris striate mosaic virus, maize streak viruses and other DNA viruses. Alternatively, Agrobacterium tumefaciens plasmid vectors may also be employed for Ti-mediated plant transformation.
Vectors, as mentioned above, may contain affinity tag sequences (hexahistidine, other metal affinity tags, streptavidin, specific epitope markers for antibody purification, glutathione-S-transferase, β-galactosidase and other tags which may assist in the isolation and purification of expressed proteins) and multiple cloning site linker sequences to assist in the cloning and purification of the protein of interest. DNA or RNA viral vectors may also contain a nucleic acid sequence coding for a signal peptide in order to direct expression of the foreign protein for secretion into interstitial fluid or the culture medium. This may simplify and enhance purification efforts due to the limited amount of endogenous proteins secreted into the interstitial fluid compartment by the plant host. An example of this may include incorporation or ligation of the sequence coding for the rice alpha-amylase signal peptide, which directs secretion of the chimeric protein into the interstitial space of the infected leaf or other plant component transfected.
In addition to plant viral vectors for plant transformation and subsequent expression and purification, mammalian or prokaryotic expression vectors may be employed for subsequent transfection or transformation into a prokaryotic or mammalian host. A preferred embodiment may be a dual mammalian/E. coli expression vector capable of transcription and subsequent expression in both bacterial and mammalian hosts. An example of this is the expression vector MEV (Mammalian Expression Vector), which contains a polylinker site with traditional restriction enzyme cloning sites (BamHI, EcoRI, SmaI, NotI, etc.), as well as SapI/EarI cloning sites. The mammalian CMV immediate-early enhancer promoter unit is located upstream and separated by an intron from the bacterial promoter unit. A Shine-Dalgarno/Kozak sequence is included for efficient expression. A histidine-tag coding sequence for efficient isolation and purification of expressed proteins is also included, which is expressed in E. coli only due to the presence of SupE/F sites.
Vectors may be constructed to allow simultaneous insertion of a nucleic acid insert into a plurality of vectors for testing in different systems. For example, vectors which are capable of expression in mammalian, bacterial and plant systems may contain the same restriction enzyme sites in the linker region of the vector DNA. Thus, a cDNA insert may be cloned into corresponding restriction enzyme sites in several different vectors, such as MEV and GENEWARE vector, simultaneously, ensuring identical frame placement of all vectors for a given cDNA insert.
As with plant viral vectors, it may also be desirable to incorporate affinity tag coding sequences (hexa-histidine, other metal tags, streptavidin, protein A, calmodulin binding protein (CBP), chitin binding domain (CBD), specific epitope markers for antibody purification, and other tags which may assist in the isolation and purification of expressed proteins) and multiple cloning site linker sequences for insertion and purification purposes into other vector DNA. Signal peptide sequences which direct the secretion of the expressed protein for packaging and subsequent secretion into the extracellular fluid matrix or culture medium may also be utilized for simplifying and enhancing purification of the expressed protein. In addition, other gene sequences which enhance the function of the vector package may also be incorporated into the vector sequence. An example of this is the incorporation of the gene sequence encoding granulocyte/macrophage-colony stimulating factor (GM-CSF) into the mammalian expression vector for proteins that may be used in the generation of antibodies or other immune responses (e.g. vaccines). GM-CSF recruits antigen presenting cells (APC; dendritic cells and macrophages), as well as enhances production of stem cell growth factors. This may result in the stimulation of the immunomodulatory system, which may increase the ability of a mammalian host to produce antibodies of higher specificity and affinity.
Affinity tags may also be used to isolate protein complexes bound to the tagged protein of interest. For example, a tandem affinity purification (TAP) tag system, previously demonstrated in yeast (Rigaut et al., 1999 Nature Biotechnology 17, 1030-1032; Gavin et al., 2002 Nature 415, 141-147), may be used to isolate proteomes, whereby the protein of interest contains the TAP tag. In the TAP system, the protein of interest is attached to two affinity markers (e.g. protein A and CBP) separated by a specific TEV protease cleavage sequence. In order to achieve expression of the protein of interest at a natural level in the yeast system, a DNA cassette encoding the TAP tag is integrated by homologous recombination into the genome of a haploid yeast cell in frame with the protein of interest.
The TAP system consists of a two-step purification system to decrease non-specific binding. The affinity purification systems, combined with the presence of the specific TEV protease cleavage sequence, also allow mild elution conditions, increasing the chances of isolating proteomes or protein complexes. Typically, a TAP purification consists first of attaching in frame a TAP gene cassette, containing the coding sequences for two affinity markers separated by the specific TEV protease cleavage sequence, onto the end of a gene sequence of interest. The TAP gene cassette may be attached to the end of a protein coding sequence by PCR cloning and amplification or by insertion and ligation into a suitable vector containing the protein of interest. The TAP-tagged protein coding sequence of interest is then inserted into a host cell, expressed, and proteins associated with the protein of interest isolated and identified. Alternatively, the TAP gene cassette may also be attached in vivo to the protein of interest by homologous recombination in frame within the chromosome of the host organism. The TAP-tagged protein sequence of interest is then expressed in vivo and associated proteins isolated.
Isolation of associated proteins is through a two-step purification procedure. A first affinity purification is performed to initially isolate any proteins associated with the TAP-tagged protein of interest. The proteome or protein complex is eluted from the first affinity purification matrix by cleavage with TEV protease, allowing a mild elution from the affinity matrix. In order to remove any non-specific proteins, contaminants and TEV protease, a second affinity purification is performed using a second affinity purification matrix. The associated proteins are then released from the bound protein of interest using EGTA elution. The isolated proteins are further isolated using denaturing gel electrophoresis. The individual protein bands are digested with trypsin and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The proteins may be identified by known database search algorithms such as Profound™ and Protein Prospector™ against databases such as NCBI SWISS-PROT or other databases known to those of skill in the art, and analyzed for protein content within the proteomes as well as between different isolated proteome structures.
Other mammalian, prokaryotic, insect, fungi or yeast vector may also be used in conjunction with the methods and compositions disclosed herein. These may include, but are not limited to, pBluescript, pCDNA3.1, pHAT, pIRES, pGBKT7, pVPack, pCMV-tag, pDual-GC, pBk-CMV, pIB-E, pMelBac, plueBac4.5/V5-His, pYD1, pPIC9K, pYES2, pIB/V5-His, pIZT/V5-His, pIZ/V5-His, pNMT1, pPICZ, pNMTsl, pMET, pPIC3JK, pGAPZ, pAO815, as well as other vectors which incorporate genetic elements necessary for expression in prokaryotic, mammalian, yeast, fungi or insect cell systems or a combination of genetic elements from different systems which allow expression in at least one of the expression systems above.
Screening of Recombinant Vectors
Transcription Analysis
Prior to a scaled-up expression of the protein of interest, vectors containing the sequence of interest may be evaluated for correct transcription of targets. Correct insertion of cDNA's into cloning vectors may be evaluated using an in vitro, prokaryotic, eukaryotic or plant transcription system in an array format, followed by size analysis of transcripts produced. A preferred embodiment is seen in
Other transcription systems may be used for evaluation of successful transcript production in each cloned cDNA vector. These may include SP6 transcription, T3 RNA polymerase or any other transcription system. For each system, the appropriate promoters (SP6 and T3 promoters, respectively) are necessary for recognition by the RNA polymerase. After addition of the RNA polymerase and transcription, the transcripts may be analyzed by polyacylamide, agarose or other gel electrophoresis for appropriate size transcripts present.
Expression Analysis
After confirmation of correct insertion of the sequence of interest into a vector, evaluation of protein expression may occur to determine the optimal vector and conditions for protein expression. Alternatively, vector constructs may be tested directly for protein expression, bypassing any transcription or RNA analysis. In all formats, evaluation of protein expression at a small scale may be used as a screening methodology to determine the optimal protein expression system for use with the described protein purification methodology.
Evaluation of protein expression may occur in a variety of systems, including plants, bacteria, yeast, fungi, insect and mammalian systems. A preferred system is the use of a plant expression system for expression of the protein of interest. However, as mentioned above, some proteins may not express well or at all in a plant expression system. Other systems may also be convenient for expression purposes, depending upon the type of equipment available for culture and amplification of the host system. Alternative embodiments for testing of the vector and inserts include bacterial, fungi, yeast, insect and mammalian systems.
Protein expression in plants may be evaluated in a variety of ways. A preferred embodiment of the invention is to evaluate protein expression in both protoplasts cultures represented at step S130 and intact plants at step S125, as depicted in
It may be desirable, instead of infecting intact plants, to transfect plant cell cultures, or protoplasts, with the plant vectors for protein expression S130 (
Protoplast suspensions may be aliquoted into microtiter plates (in this example, a 2×3 microtiter plate array, but other microtiter plate formats can be utilized) after enzymatic digestion, washing and culture using an appropriate medium, preferably in duplicate. A preferred embodiment may utilize commercially available basal Murashige and Skoog medium, although other medium preparations which provide a balanced mixture of macro and micro-elements, soluble carbon sources, nitrogen vitamins and other growth factors necessary for maintenance of protoplasts in vitro. It is well known to those of ordinary skill in the art that many different combinations and ranges of media constituents can be used successfully for protoplast expansion.
After suspension of protoplasts and subsequent incubation in a suitable medium, protoplast cells may be transfected with the DNA or RNA using a variety of methods. Preferably, the gene of interest is incorporated into a GENEWARE® vector, is packaged or encapsidated and the encapsidated virus is used to infect protoplasts. In this way, protoplasts are transiently transfected with a suitable vector and induced in vitro to express the desired protein. Direct DNA microinjection, electroporation, liposome carriers, particle bombardment (biolistics), silicon carbide fibers or other methods may also be used to introduce and express foreign genes in plant cells.
Alternatively, Agrobacterium tumefaciens-mediated Ti transfer of cloned DNA may also be used to introduce and express foreign genes in plant cells. For example, cloned DNA may be inserted into a suitable vector which is taken up by Agrobacterium. The protoplasts or intact plants are then incubated in the presence of the DNA-containing Agrobacterium. Agrobacterium, through the presence of the Ti gene, mediates the transformation and integration of the insert DNA into the plant cell host. Protein expression may subsequently be induced under the control of inducible promoters co-transfected with the DNA of interest, or natural promoters may be transfected which may subsequently place control of expression under the plant host. Such natural promoters may include constitutively active promoters, which may be modified to express foreign genes at high levels.
Protoplasts may also be used as an initial screening tool for determining if an intracellular or secretory pathway is used for protein expression. Microwell culture plates are first centrifuged to separate protoplast cells from cell culture media. The media is aspirated and collected for parallel purification along with protoplast cell lysate. Both the protoplast cell lysate and media, as well as intact plant homogenate suspensions, are added to separate wells in a 96-well filter plate containing metal binding matrices, such as Ni-NTA beads or Ni-chelating disks (Swell-Gel, Pierce). The flow through fraction is discarded, and the metal binding matrix (such as Ni-agarose) washed with 40 mM Imidazole/0.5 M NaCl/Phosphate buffer (pH 7.9). The bound proteins are then eluted with 1 M Imidazole/0.5 M NaCl/Phosphate buffer (pH 7.9) and analyzed on 1-D or 2-D polyacrylamide gels (step S140 in
In addition to identification on 1-D gels, target bands may also be excised from the 1-D polyacrylamide gels and analysed by tryptic MALDI-TOF (Matrix Assisted Laser Desorption/Ioniazation-Time of Flight Mass Spectrometry) which may ensure correct insertion of the cDNA into the vector (correct reading frame) and confirm correct protein expression. Tryptic MALDI may be performed by first eluting protein from the polyacrylamide gel, followed by trypsin digestion of the proteins, purification of the fragments, lyophillization and subsequent solubilization in the proper solvent. The sample is then analyzed using MALDI-TOF Mass Spectrometry or any other ion desorption method allowing sequential peptide cleavage and mass measurements. As an alternative embodiment, target bands may be excised from the 1-D polyacrylamide gels or transferred to nitrocellulose or PVDF membranes and eluted from the membranes. The isolated protein band can then be sequenced using standard protein sequencing techniques (e.g. Edman degradation or any other protein sequence method). In addition, trypsin digestion may also be performed on the isolated protein, after which standard protein sequencing techniques are applied (e.g. Edman degradation or any other protein sequence method).
After analysis on 1-D polyacrylamide gels and MALDI-TOF MS, the probability that the protein expressed is the correct protein may be calculated using standard database analysis.
In a manner similar to that described above with respect to
Vectors and their insert may also be evaluated using bacterial, fungi, yeast, insect and mammalian systems. For example, in situations where no protein is expressed from transfected protoplast cultures or infected plants, the corresponding MEV cDNA clone may be analyzed for expression in E. coli to assure that DNA transfection error into plants or protoplasts is not the cause of the lack of protein expression. Alternatively, bacterial, fungi, yeast or insect may be tested in parallel with a plant expression system to determine the optimal system for protein expression and purification.
There are many methods known to one of ordinary skill in the art for expressing foreign proteins from a cDNA vector in a prokaryotic host. A preferred embodiment may include the transformation of a suitable host strain of E. coli, such as NovaBlue DE3, with MEV vector containing cDNA or genomic DNA inserts in a 96-well format. The transformants may be plated on solid media with selective antibiotics, depending on the vector used, and grown overnight in a deep 96-well block at 37° C. After overnight growth, the E. coli cultures may be diluted into fresh media containing isothiopropyl galactoside (IPTG) to induce expression of the protein through the β-galactosidase promoter in the vector. Alternatively, other strains of E. coli or suitable prokaryotic host strain may be used for propagating vector DNA and their inserts, as well as other vectors with alternative inducible promoter systems, such as temperature-dependent expression or other inducible systems.
After logarithmic growth for a defined period of time, 2 microliters of culture may be spotted onto a nitrocellulose membrane in an 8×12 grid, and a Western blot may be performed using antibody to the target protein or tag. Alternatively, the expressed protein, with its attached hexahistidine tag, may be isolated and purified as above on a SwellGel Ni chelating matrix in a 96-well filter plate format. The eluted protein may be analyzed on 1-D polyacrylamide gels for determination of proper size expression. In addition, tryptic MALDI-TOF may be performed on excised protein bands for further identification.
Protein Expression Scale-Up
After protein evaluation and screening, a larger scale protein expression and purification may be commenced. The evaluation of the protein, as is indicated at S3 in
The steps depicted in
The tobacco plants are then harvested, and disintegrated in, for instance, a Waring blender or commercial juicer to release the desired protein or proteins from the cells of the leaves in the form of green juice, as indicated at S11 in
After being treated with a clarifying agent, the green juice may be further processed in one of at least two alternative manners. First, as depicted at S13 in
Alternatively, after step S12, the PEG treated green juice may be subjected to centrifugation at a force of 3,700 G for approximately 20 minutes in order to separate the larger protein aggregate from the clarified green juice, as indicated at step S14 in
If necessary, the clarified green juice may also be subjected to a freeze and thaw as is indicated at S15 in
The volume of the clarified green juice is next normalized such that a plurality of samples containing diverse proteins can be simultaneously purified. During normalization, urea or glycerol may be added to predetermined concentrations and/or pH adjustment of the sample may occur. For example, urea at concentrations ranging from 50 mM to 4 M and glycerol at concentrations ranging from 5% w/v to 50% w/v may be employed and NaOH (sodium hydroxide) or a sodium phosphate or Tris buffer, may be used to raise pH from 7.2-7.3 to 7.5-8.0. It should be understood that during normalization, only pH adjustment may occur. Levels of urea and glycol may or may not be included depending upon the characteristics and properties of the desired protein of interest.
The normalized clarified green juices are then loaded into a purification apparatus, such as the apparatus described below with respect to
Further description of the methods of the present invention depicted in
As shown in
The feed reservoir 5 is connected to a tube 15 that is connected to a first valve 20. The first valve 20 is connected to a tube 25 that is further connected to a pump 30. The pump 30 may be any of a variety of pumps, but is preferably a low velocity pump that moves the clarified green juice through the purification apparatus of the present invention at a generally slow rate. For instance, the pump 30 may be a peristaltic pump such as a variable speed pump manufactured by ISMATEC® with a flow range of 0.01 to 44.4 mL/minute. Such pumps are also multi-channel pumps enabling simultaneous purification of multiple proteins, each in its own purification apparatus in a manner described in greater detail below with respect to
The pump 30 is connected to a second valve 40, which is in turn connected to tube 45, which is connected to a column 50. The column 50 is connected to tubing 55 that is connected to a third valve 60. The third valve 60 is connected to a tube 65 that is connected to a flow-through collection reservoir 70. It should be understood from the following description that clarified green juice loaded into the feed reservoir 5 is transported via pumping action of the pump 30 from the feed reservoir 5, through the various tubes 15, 25, 35, 45, 55 and 65 and through the column 50 and valves 20, 40 and 60, into the collection reservoir 70.
The column 50 possesses a porous frit that retains a material therein, but allows the flow of fluid therethrough such that there can be contact and potential interaction between the flowing fluid and the retained material. In the purification apparatus of the present invention, the material in the column 50 is, for instance, an affinity resin, such as those marketed by Qiagen®, or other similar material for temporarily retaining the desired protein of interest. As the clarified green juice flows through the column 50 the protein of interest is attracted to and retained on the affinity resin.
The valves 20, 40 and 60 are connected to tubes 75, 80 and 85, respectively and are included in the purification apparatus for a variety of purposes. In purification mode, the valve 20 is set to allow fluid communication (fluid flow) from the tube 15 to the tube 25. The valve 20 may also be set to allow fluid flow from the tube 75 into the tube 25 for cleaning purposes, for removal of the purified protein of interest (as is described further below), or for priming the pump 30 and system equilibration, among other functions. The valve 20 may also be set to allow fluid communication between the tube 15 and 75.
In purification mode, the valve 40 is set to allow fluid communication between the tube 35 and the tube 45. However, the valve 40 may be set to allow fluid flow between the tube 35 and the tube 80 for cleaning or priming the pump 30, or the valve 40 may be set to allow fluid flow between the tube 80 and the tube 45 for washing the column 50 or for removal of the purified protein of interest.
In purification mode, the valve 60 is typically set to allow fluid communication between the tube 55 and the tube 65. The valve 60 may also be set to allow fluid communication between the tube 55 and tube 85 to allow for washing of the column 50 or for removal of the isolated protein of interest in the column 50. The valve 60 may also be set to allow fluid communication between the tube 85 and 65 to permit flushing and cleaning of the tube 65.
It should be understood that the valve 40 is optional and may alternatively be omitted from the apparatus depicted in
Under some circumstances, the system may need to be primed. Specifically, fluid may be introduced from receptacle 100 to line 15, line 25, pump 30, line 35, line 45 and line 55 by manipulation of the valve 20 and 85. Typically, the system would be primed with the column 50 removed, and lines 45 and 55 directly connected to one another. After the system has been primed, the removable column 50 is re-inserted between lines 45 and 55, as shown in
For operation of the purification system, clarified green juice is put into the juice receptacle 5. Thereafter, the pump 30 is operated to draw clarified green juice out of the juice receptacle 5, into the tube 15, through the valve 20 and of course the pump 30, through tubes 35 and 45 and valve 40 and into the column 50. In the column 50, the clarified green juice interacts with the material disposed in the column 50, and ideally, all protein of interested is retained within the column 50 while the remainder of the clarified green juice flows out of the column 50, basically as waste. The waste juice passes through the tubes 55 and 65 and valve 85 and into the collection reservoir 70.
Returning now to
Thereafter, the valves 20 and 60 are set for purification mode and the clarified green juice and buffer solution mixture are pumped from the receptacle 5, through the column 50 and into the collection reservoir 70, as is depicted in
After all of the mixture has passed through the column 50, contaminates must be washed out and certain buffer components, e.g. PEG and urea removed, as indicated at step S20 in
Next, as indicated in step S21 in
Alternatively, prior to elution from column 50, the protein of interest may be re-folded in situ on the column matrix through the introduction of a linear gradient of renaturation buffer (e.g. phosphate buffered saline, tris buffered saline or other buffers used in renaturation) after washing. For example, many histidine tagged proteins are purified under denaturing conditions, exposing the histidine tag at either the carboxy or amino terminus, thereby increasing binding of the tag to binding groups present on the metal affinity matrix. The histidine-tagged proteins are then subsequently eluted in their denatured state from the metal affinity matrix by lowering the pH of the buffer passing through the column or introducing a high concentration of imidazole or EDTA. The eluted proteins, especially at higher concentrations, sometimes fall, or precipitate, out of solution. This may be caused by intermolecular interactions between hydrophobic groups which are exposed due to the denatured state of the eluted protein. If the proteins cannot be resolubilized, the overall yield of protein is decreased. However, by the introduction of a linear gradient of renaturation buffer after washing, the protein may be allowed to re-fold while bound to the affinity matrix. Upon re-folding, the previously exposed hydrophobic groups are shielded, preventing intermolecular hydrophobic interactions and precipitation of the proteins.
It is important that a gradient is employed for inducing re-folding of the protein. Although practice of the claimed methods is not dependent upon an understanding of the mechanism of the invention, it is believed that the gradual introduction of renaturation buffer assists in the proper folding of the protein while bound to the affinity matrix, giving the bound protein time to properly re-fold into complex tertiary or quartenary structures. After the re-folding of the protein of interest on the column, the protein may now be eluted from the column with the introduction of elution buffer.
Linear gradient makers may be used where re-folding of the protein of interest in situ while bound to the affinity matrix is desired. Linear gradient makers allow the gradual introduction of the renaturation buffer over a set volume or period of time. Linear gradient makers may employ at least one pump or proportioning valve for drawing from two reservoirs containing the starting and final buffer, such as reservoirs 105 and 110 depicted in
Gradual introduction of the renaturation buffer may occur by stepwise, instead of a linear gradient, introduction of renaturation buffer. For example, buffer solutions of decreasing salt or urea concentrations may be flowed over the column in a stepwise fashion. It is appreciated that one of ordinary skill in the art will appreciate the many ways by which a gradual introduction of renaturation buffer may take place to re-fold a denatured protein of interest in situ on the affinity matrix.
There are groups of proteins that are difficult to separate from one another. Therefore, in an alternate embodiment depicted in
The valve 48 is further connected to a tube 90. In purification mode, the valve 48 is set to direct flow of juice from the tube 47 to the tube 49 and into the column 50. However, the valve 48 may further be set to allow fluid communication between the tube 47 and tube 90. As well the valve 48 may be set to allow fluid communication between the tube 90 and tube 49 for cleaning purposes, flushing purposes or for removal of purified protein in a manner described in greater detail below.
Further, as shown in
As shown in
The apparatus in
The computer depicted in
The computer depicted in
In accordance with the present invention, it is possible to purify quantities of proteins measured in milligrams in a cost effective and efficient manner. Other methods and apparatus allow for extremely large quantities (measured in 100 g to Kg) or extremely small quantities (measured in μs), therefore the methods and apparatuses of the present invention fulfill a need.
A subset of proteins, with emphasis on markers whose expression is restricted to either the lung or the brain, were selected for GENEWARE®-based expression and subsequent purification in parallel. Protein databases such as the Human Protein Index (HPI) and SWISS-PROT were screened for potential proteins and a subset chosen based on the availability of full-length clones from both in-house and commercially available gene collections. Each full-length clone was assigned a sequence ID (SeqID) to permit tracking of the DNA sequence and resulting protein in the laboratory information management system (LIMS), from vector generation through to confirmation (
For screening, sufficient in-vitro transcript was generated for each clone to inoculate three 21-day old Nicotiana benthamiana plants. Twelve to fourteen days after inoculation, the plant material above the inoculated leaves was harvested, weighed and macerated to obtain a green juice. In a deep-well block (96 well), one volume green juice was combined with 2 volumes extraction buffer (25 mM Tris pH 8.0, 500 mM NaCl, 2 mM PMSF, 7 mM β-mercaptoethanol) and adjusted to 4% w/v PEG (1500 ul final volume), to simulate the extract obtained during protein production. After storage for half an hour at 4° C., the green juice was centrifuged at 3000×G for 20 minutes to obtain a clarified green juice, containing the target protein. To capture the target protein, 700 ul of the clarified green juice was combined with 25-ul affinity resin (Qiagen Ni-NTA) in a 96-well filter plate and incubated for one hour at room temperature. The filter was sufficiently hydrophobic to retain the clarified green juice, which could be removed following incubation, by centrifugation at 1000×G for 5 minutes. The affinity resin, with the captured protein, was retained by the filter and washed twice with 700 ul wash buffer (16 mM Tris, pH 8.0, 330 mM NaCl, 5 mM imidazole), with centrifugation at 1000 G for 5 minutes between washes. Recovery of the target protein from the affinity resin was achieved by incubating the resin in 60 ul elution buffer (16 mM Tris, pH 8.0, 150 mM NaCl, containing either 200 mM Imidazole or 200 mM EDTA) for 5 minutes and centrifuging (1000×G for 5 minutes) to recover the eluant. The elution step was repeated to yield 120 ul of final product. To assess the expression level of each tagged protein, the eluent from each purification was analyzed by SDS-PAGE. If a protein band of approximately the correct molecular weight (+/−20%) was observed following Coomassie staining, and no co-migrating bands were observed in the negative controls, successful expression of target protein was assumed. The protein level was quantified by densitometry, using a bovine serum albumin standard. This variable was inputted into the LIMS system, together with the recorded plant mass and the number of plants required to produce the target protein was determined.
For protein production, N benthamiana plants were sown in lots of nine. To facilitate tracking and inoculation, the number of plants required for each protein target was rounded up to the nearest multiple of nine. The expression level for each protein will vary greatly and subsequently so too will the number of plants required to achieve a given protein level. Lots varying from nine to ninety-six 21-day old plants were typically used and the in vitro transcription reactions scaled accordingly. Twelve to fourteen days after inoculation, the plant material above the inoculated leaves was harvested, weighed and combined with two volumes of chilled extraction buffer. The extraction buffer was vacuum infiltrated into the plant material to ensure even buffer/plant material distribution and the green juice obtained using a commercial juice extractor. PEG was added to 4% w/v and the green juice stored at 4° C. for half and hour, to permit aggregation and precipitation of the chlorophyll-containing component of the extract. The green juice was clarified by filtration, employing 4% w/v perlite as a filtration aid. The clarified green juice was adjusted to 10% v/v glycerol, to minimize hydrophobic protein interactions with the affinity resin and the extract volumes normalized with extraction buffer. Each channel of the pre-equilibrated purification apparatus was loaded with clarified green juice containing a particular target protein. In the case where the volume of green juice for a given target protein was substantially greater than for the other target proteins, the clarified green juice was divided into two of more of the channels and the purified proteins pooled following elution from the affinity resin. The clarified green juice was passed over the affinity resin and the histidine-tagged protein retained on the Ni-NTA affinity resin. Contaminating plant proteins were removed by passing 10 column volumes of wash buffer over the column and the target protein recovered using an elution buffer containing 200 mM EDTA. The composition of the extraction buffer, wash buffer and elution buffer were identical to those employed in the screening step. Aliquots of each eluant were analyzed by SDS-PAGE and densitometry of the Coomassie-stained protein bands performed, to determine the concentration of the protein. Where necessary the proteins were concentrated by ultrafiltration and all proteins were dialyzed into phosphate buffered saline, prior to storage at −20° C. SDS-PAGE was performed on the final concentrated and dialyzed proteins, to determine protein purity and tryptic MALDI was performed to confirm protein identity.
Table 1 summarizes the results for production runs were between 5 and 15 unique proteins were expressed using GENEWARE® and purified in parallel. Based on the screening, sufficient plants were inoculated to obtain 1.5 mg of purified protein, with a minimum of 9 plants per target protein. In production mode the required protein level was achieved or exceeded for 10 of the 27 targets. In the case of 11 targets a second round of production with appropriately adjusted plant numbers would be performed to meet the protein requirement. For the six targets were no protein was recovered, GENEWARE® expression on a 9-plant lot would be performed to confirm the result. If no protein is recovered following this purification, the SeqIDs are identified as incompatible with GENEWARE® and evaluated in another expression system e.g. mammalian.
Various details of the invention may be changed without departing from its or its scope. Furthermore, the foregoing description of the embodiments according resent invention is provided for the purpose of illustration only, and not for the e of limiting the invention as defined by the appended claims and their equivalents.
This application claims priority to U.S. provisional application 60/338,725, filed Dec. 5, 2001, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60338725 | Dec 2001 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10309756 | Dec 2002 | US |
| Child | 11303548 | Dec 2005 | US |
