WO2006091892A2

WO2006091892A2 - Methods of producing recombinant proteins

Info

Publication number: WO2006091892A2
Application number: PCT/US2006/006741
Authority: WO
Inventors: Alan Greener; Marie Callahan; Xiaodong Wang
Original assignee: Gene Therapy Systems, Inc.
Priority date: 2005-02-25
Filing date: 2006-02-23
Publication date: 2006-08-31
Also published as: WO2006091892A3

Abstract

Described herein are methods of producing a recombinant protein. The methods can include providing a host cell comprising a recombinant nucleic acid construct, wherein said recombinant nucleic acid construct encodes a recombinant protein and contacting the host cell with a first compound to which the host cell is sensitive. The methods can also include harvesting the host cells and recombinant protein from said host cell. The methods can be practiced with in vitro systems as well. Also described are systems and kits related to the methods. The methods systems and kits can be used to produce soluble recombinant protein and to increase the level of a recombinant protein relative to cellular protein in a host cell

Description

METHODS OF PRODUCING RECOMBINANT PROTEINS

BACKGROUND OF THE INVENTION Related Cases

[0001] The present application claims priority to U.S. Provisional Application Serial No. 60/710,859, filed on August 24, 2005, and U.S. Provisional Application Serial No. 60/656,649, filed on February 25, 2005, both of which are incorporated herein by reference in their entirety. Field of the Invention

[0002] Described herein are methods of enriching the level of recombinant protein relative to native host cell proteins in host cells, as well as methods of producing soluble protein, increasing the level of soluble recombinant proteins in host cells and increasing the specific activity of the recombinant proteins produced. Also described are kits comprising a compound to which a host cell is sensitive, which compound causes an increase in the ratio of expression of recombinant protein to endogenous host cell protein and kits comprising a compound to which a host cell is sensitive, that can cause an increase in the level of soluble recombinant protein compared to insoluble recombinant protein. Description of the Related Art

[0003] The advent of recombinant DNA technology has made it possible to engineer host cells that over-express proteins of interest. A goal of recombinant protein technology is to produce large quantities of protein that retain the biological activity of the naturally-occurring protein of interest. Cloned genes are commonly introduced into bacterial, yeast, mammalian and insect cells to achieve expression of the encoded protein. As one example, prokaryotic gene expression systems represent a widely used mechanism for producing a protein of interest. Prokaryotic host cells are particularly attractive host cells for recombinant protein expression due to the fact that many bacteria have a rapid doubling time and are inexpensive to propagate. Furthermore, for several prokaryotes, many commercially available vectors exist that enable the researcher to control the timing and level of expression of the recombinant protein of interest. In many cases recombinant proteins exceed 30% of the total prokaryotic host cell protein content. In addition to the prokaryotic systems, yeast, mammalian, and insect systems, and even in vitro systems have been used to produce recombinant proteins.

[0004] There is still a need to improve the various systems and methods for producing recombinant proteins. In particular, improved methods and systems are needed that provide better yield, purity, and quality of the recombinant protein.

[0005] Several approaches have been taken to improve the yield and purity of recombinant proteins, while not compromising the biological activity of the protein. One set of approaches focused on increasing the amount of recombinant protein expressed in the host cell. Methods that have met with some success have included, for example, increasing the number of copies of the DNA encoding the recombinant protein in the cell by using expression plasmids that are present within the host cells in high copy numbers. See, U.S. Patent No. 5,716,803. Another approach has been to increase the efficiency of transcription of the recombinant DNA by cloning the gene encoding the recombinant protein under the control of promoters and transcription regulatory elements that are capable of yielding high levels of transcription. Additionally, research efforts have focused on increasing the stability of recombinant mRNA's. See, EP Publication 0204401. Yet other efforts have focused on increasing the efficiency of translation of recombinant mRNA's into proteins. See, U.S. Publication 2004/0260060.

[0006] Another set of approaches to improve recombinant protein technology involved engineering recombinant proteins to facilitate their purification away from endogenous host cell proteins. These approaches involve cloning the DNA encoding the protein of interest so as to produce a fusion between the protein of interest and another "tag" that specifically binds to another compound ("binding partner"). The tag portion of the fusion protein can be exploited in affinity chromatography to purify the recombinant fusion protein away from endogenous host cell proteins. A major drawback of this technique is that the activity of the recombinant protein is often affected by the modification of adding a tag to the protein. Besides possibly changing the biochemical properties of the protein, the purification often involves harsh elution conditions that can compromise its biological activity. Another common drawback arises because endogenous host cell proteins may have varying degrees of affinity for the same binding partner as the tag. Depending on the degree of affinity of the host cell proteins for the binding partner, endogenous host cell proteins will co-purify with the tagged protein of interest. The higher the concentration of endogenous host cell proteins, the more likely it is that the undesired host cell proteins will co-purify with the recombinant protein of interest.

[0007] Also, efforts have focused on avoiding the formation of insoluble protein aggregates that are problematic with many of the existing expression systems and protocols. For example, frequently, over-expression of many recombinant proteins yields products which fold incorrectly in the cell, forming insoluble aggregates, including, for example aggregates that are called inclusion bodies. See Williams et at, (1982), Science 215:687-688 and Schoner et al, (1985), Biotechnology 3:151-154; both of which are incorporated herein by reference in their entirety. Some factors which have been suggested to be involved in the formation of inclusion bodies include the high local concentration of protein; a reducing environment in the cytoplasm of prokaryotes that prevents the formation of disulfide bonds; lack of post- translational modifications to eukaryotic proteins expressed in prokaryotic hosts; and the inability of recombinant proteins, and particularly recombinantly expressed eukaryotic proteins, to interact with chaperones and other prokaryotic enzymes involved in protein folding. Depending on the organism from which the gene has been isolated, the percentage of recombinant proteins that form primarily or exclusively insoluble (and thus inactive) products can vary from 40% - 80%.

[0008] Depending upon the purpose for which the recombinant protein is to be used, solubility can be a critical factor. Although solubility by itself does not guarantee that the protein is correctly folded, proteins that are insoluble are by definition incorrectly folded and would most likely exhibit a significant reduction in specific activity. If the protein is to be crystallized, used in an in vitro biological system, or transfected directly into cells, for example, proper folding is essential. Although the fact that a protein is soluble does not necessarily guarantee that the protein retains its in vivo activity, insoluble protein - by definition - will not retain its in vivo activity.

[0009] One approach to address the problem of insolubility of recombinantly expressed proteins in prokaryotes has been to engineer host cells that over-express proteins involved in proper protein folding, such as chaperones. See, e.g., U.S. Patent Application No. ^' 10/500,883, which is incorporated herein by reference in its entirety. The over- expression of chaperones and simultaneous over-expression of a protein which in the absence of the chaperones leads to inclusion body formation was found to substantially decrease the amount of inclusion body formed in some cases. However, these results are often empirical, and depend on the individual chaperone/recombinant protein pairing involved.

[0010] Another approach to the problem of recombinant protein solubility capitalizes on the discovery that in some cases, short peptide leader sequences facilitate correct folding of the protein. Recombinant protein expression systems have been designed to create fusion proteins with the protein of interest and a short peptide leader sequence fused to the N-terminus of the protein. Sec, e.g., U.S. Patent Serial No. 10/997,535, which is incorporated herein by reference in its entirety. Drawbacks to this approach are that the success rate is low and it requires recloning the gene encoding the protein of interest into a new vector. Furthermore, often the presence of the additional amino acids is undesirable. While some leader sequences are designed such that they can be removed by proteolytic digestion, such processes are not 100% efficient.

[0011] A different approach involves attempting to solubilize insoluble proteins. For example, insoluble aggregates of proteins, such as inclusions bodies, have been treated with powerful denaturing agents such as high concentrations of urea and guanidium hydrochloride. Frequently, however, the proteins re-aggregate during the renaturation process. Thus, this technique has a low success rate and has to be adjusted for every different recombinant protein tested.

[0012] It is apparent that there is still much room for improvement related to recombinant protein systems. The methods and kits described herein overcome many of the above-described drawbacks.

SUMMARY OF THE INVENTION

[0013] Some embodiments relate to methods, systems, and kits for producing recombinant protein. For example, the methods, systems and kits can be used with prokaryotic, eukaryotic, or in vitro expression systems. The methods, systems, and kits can include the use of a compound to which the cell or system is sensitive. For example the compound can be an antibiotic to which the recombinant host cell/expression system is sensitive or not resistant. The recombinant host cell or in vitro expression system can be contacted with the compound and the recombinant protein can be obtained from the system. The methods, systems and kits can be utilized, for example, to produce soluble recombinant protein and to produce recombinant protein in a greater ratio compared to host cell protein.

{0014] One embodiment relates to recombinant protein expression in a host cell, wherein the host cell is sensitive to a first compound. The host cell can include a recombinant nucleic acid that encodes a recombinant protein. The host cell can be contacted with the first compound, and the recombinant protein can be obtained from the host cell.

[0015] In some embodiments, the recombinant protein obtained from the host cell can be soluble, and the amount of soluble protein obtained from the host cell contacted with the first compound can be greater than the amount of soluble protein that is obtainable from the host cell when the host cell is not contacted with the first compound. In other embodiments, the amount of recombinant protein present in host cells that have been contacted with the first compound increases relative to the total level of protein within the host cell, when compared to host cells that have not been contacted with the first compound.

[0016] In some embodiments of the methods above, the host cells can be prokaryotic, and in other embodiments of the methods above, the host cells can be eukaryotic. For example, in some embodiments, the host cell can be a Gram negative bacterium, such as Escherichia coli, Salmonella typhimurium or a member of the genus Pseudomonas, or a Gram positive bacterium, such as a member of the genus Bacillus. Exemplary E. coli host cell strains useful in the above embodiments include E. coli BL21, BL21(DE3), DH5α, JM109, JMlOl, HBlOl, BL21(DE3) pLysS, BL21(DE3) pLysE, DHlOB, XL-I Blue, XL-10 Gold, NM522, and TOPlO. In some embodiments, the host cells of the methods described herein can exclude one or more of the host cells enumerated herein.

[0017] In other embodiments, the host cell can be eukaryotic. For example, some embodiments of the methods above provide yeast host cells such as Saccharomyces cerevisiae, Pichia pastoris or Schizosaccharomycetes pombe. Other embodiments provide mammalian host cells such as for example CHO, COS-7, 293, or NIH-3T3 cells and the like. Yet other embodiments provide insect host cells, such as Sf-9 cells. In some embodiments, the host cells of the methods described herein can exclude one or more of the host cells enumerated herein.

[0018] In some embodiments, the first compound can be an antibiotic or an analog of an antibiotic. For example, in some embodiments, the first compound can be streptomycin, gentamycin, amikacin, kanamycin, neomycin, netilmycin, paromycin, spectinomycin, tobramycin, erythromycin, azithromycin, clarithromycin, roxithromycin, telithromycin, chloramphenicol, puromycin, cycloheximide, diphtheria toxin, G-418, hygromycin, blasticin S, doxorubicin or the like. In some embodiments, the first compound of the methods described herein can exclude one or more of the compounds listed above.

[0019] In some embodiments, the host cell can be contacted with a first compound that inhibits translation. For example, in some embodiments, host cells can be contacted with a first compound that targets the small ribosomal subunit, such as an aminogylcoside in embodiments with prokaryotic host cells. In other embodiments, the first compound can target the large ribosomal subunit.

[0020] In embodiments of the methods above, the host cells can be contacted with a sublethal amount of the first compound, such as an amount of a first compound that does not immediately kill the host cell.

[0021] In some embodiments, the first compound can be administered in a single dose at one time. In other embodiments, the first compound is administered in more than one dose, at more than one time. For example, in some embodiments the host cell can be an E. coli cell and the first compound can be spectinomycin. Several doses of spectinomycin can be administered to the host cell, such that the concentration of spectinomycin to which the host cell is contacted increases from about 5μg/ml, to 10μg/ml, to 15μg/ml, to 20μg/ml, to 25μg/ml, to 30μg/ml, to 35μg/ml following the administration of each dose. The administration of each dose can be over time, for example, the first dose can be administered at approximately the same time as a regulator compound, and subsequent doses can be administered every 10, 15, 20, 30, 40, 50, or 60 minutes, for example.

[0022] In some further embodiments of the methods above, the recombinant nucleic acid construct can encode a selectable marker which enables the host cell to grow in the presence of a selection compound. The host cells can be contacted with the selection compound to ensure growth and proliferation of only those host cells having and maintaining the recombinant nucleic acid construct.

[0023] In other embodiments, the recombinant nucleic acid construct can be configured such that the nucleic acid encoding the recombinant protein can be operably linked to a heterologous promoter. In further embodiments, expression of the recombinant nucleic acid from the heterologous promoter can be induced by a regulator compound. Thus, in some embodiments, the host cell also can be contacted with the regulator compound. Accordingly, in some embodiments, the host cell is contacted with the first compound after the host cell is contacted with the regulator compound, for example, approximately 30 minutes, 60 minutes, 90 minutes 120 minutes, 180 minutes, 240 minutes, or 300 minutes after the host cell can be contacted with the regulator compound. Alternatively, the host cell is contacted with the first compound at about the same time that the host cell is contacted with the regulator compound.

[0024] In further still further embodiments, the heterologous promoter can be a bateriophage promoter, for example, and the regulator compound can induce expression from the bacteriophage promoter. For example, the bacteriophage promoter can be a phage T7 promoter. In some embodiments, the regulator compound can be IPTG, and IPTG induces expression from the T7 promoter.

[0025] In some embodiments, the host cells can be contacted with compounds other than the first, such as an alkali metal salt, for example NaCl or an alkaline metal earth salt. In some embodiments, the host cells can be contacted with osmoprotectants, such as glycine, glycinebetaine, ectoine, sugars, sugar alcohols, carnitine, proline, glutamate, sorbitol, and mannitol. In other embodiments, host cells can be contacted with oxidizing agents or antioxidants, such as hydrogen peroxide, oτ reducing agents, such as 2-mercaptoethanol or dithiothreitol.

[0026] Other embodiments relate to methods for production of a recombinant protein. An E. coli BL21(DE3) host cell that is sensitive to spectinomycin, erythromycin, chloramphenicol, kanamycin or streptomycin can be provided. The BL21(DE3) recombinant host cell harbors a recombinant nucleic acid construct encoding the recombinant protein. The host cell can be contacted with the antibiotic to which it is sensitive, and the recombinant protein can be obtained from the cell.

[0027] Yet other embodiments relate to methods for the production of a recombinant protein in vitro. An in vitro translation, or transcription translation system can be provided, as well as a recombinant nucleic acid that encodes the recombinant protein of interest. The recombinant nucleic acid encoding the recombinant protein can be operably linked to translation, or transcription and translation regulatory elements, respectively, that are recognized by the in vitro system. The in vitro system can be contacted with the recombinant nucleic acid, and the in vitro system and nucleic acid are contacted with a composition that includes a compound that reduces the rate or amount of recombinant protein synthesis from the in vitro system compared to an in vitro system and recombinant nucleic acid that have not been contacted with the composition. For example, in some embodiments, the compound can be an antibiotic or an analog of an antibiotic, such as streptomycin, gentamycin, amikacin, kanamycin, neomycin, netilmycin, paromycin, spectinomycin, tobramycin, erythromycin, azithromycin, clarithromycin, roxithromycin, telithromycin, chloramphenicol, puromycin, cyclόheximide, G-418, hygromycin, blasticin S and doxorubicin, or analogs thereof. In some embodiments, the in vitro system can be contacted with the composition before it can be contacted with the nucleic acid, and in other embodiments the in vitro system can be contacted with the composition and recombinant nucleic acid at approximately the same time.

[0028] Other embodiments relate to methods for the production of a recombinant protein. A host cell having a recombinant nucleic acid construct that operably encodes the recombinant protein can be provided. The host cell can have a selectable marker, and can be cultured in media that includes a selection compound that enables only the host cells that include the selectable marker to propagate. The host cell can be contacted with a first compound that reduces the production of native protein, and the host cells subsequently can be harvested, and the recombinant protein can be obtained therefrom.

[0029] Other embodiments relate to methods of increasing the ratio of expressed recombinant protein to expressed host cell protein. A host cell can be provided that has endogenous DNA that encodes host cell proteins. The host cell also can have a recombinant nucleic acid construct that encodes a recombinant protein, and can include a selectable marker. The host cell can be sensitive to a first compound. The host cells can be cultured in media that includes a selection compound such that only the cells having the recombinant nucleic acid construct with the selectable marker can propagate. The host cell can be contacted with the first compound. The first compound can reduce host cell protein synthesis. Contacting the host cell with the first compound can increase the ratio of expressed recombinant protein to expressed host cell protein.

[0030] Other embodiments relate to methods to increase the ratio of soluble recombinant protein to insoluble recombinant protein expressed in a host cell. A host cell having a selectable marker can be provided. The host cell can also have a recombinant nucleic acid construct that provides for expression of a recombinant protein within the host cell. The host cell can be also sensitive to a first compound, which reduces host cell protein synthesis. The host cell can be cultured in a media with a selection compound that enables only host cells comprising the selectable marker to propagate. The host cell can be contacted with the first compound, which can result in an increase in the ratio of soluble recombinant protein to expressed to insoluble recombinant protein expressed in the host cell.

[0031] Other embodiments relate to an improved method of producing a recombinant protein from a recombinant nucleic acid construct in a host cell, wherein the host cell can be grown in media including a selection compound, and wherein the host cell can include a selectable marker. The improvement can include reducing the level of background protein expression by administering to the host cell prior to harvesting the recombinant protein an amount, such as for example a sublethal concentration, of a first compound to which the host cell is sensitive, wherein the first compound is a reducer of native protein synthesis by said host cell.

[0032] Other embodiments relate to improved methods for producing a recombinant protein from a recombinant nucleic acid construct in a host cell. The host cell can include a selectable marker, and can be grown in media that includes a selection compound. The improvement can include increasing the level of soluble recombinant protein expressed, by administering to the host cell prior to harvesting the recombinant protein an amount, for example a sublethal concentration, of a first compound to which the host cell is sensitive, wherein the first compound is a reducer of native protein synthesis by said host cell.

[0033] Additional embodiments relate to improved methods for producing recombinant protein with increased specific activity. A host cell having a selectable marker can be provided. The host cell can also have a recombinant nucleic acid construct that provides for expression of a recombinant protein within the host cell. The host cell can be also sensitive to a first compound, which reduces host cell protein synthesis. The host cell can be cultured in a media with a selection compound that enables only host cells comprising the selectable marker to propagate. The host cell can be contacted with the first compound, which can result in an increase in the specific activity of the recombinant protein compared to the specific activity of recombinant protein from cell cultures that have not been contacted with the first compound..

[0034] Yet other embodiments relate to methods of increasing the level of a recombinant protein relative to the level of cellular protein in a host cell. The host cell can be contacted with a regulator compound that induces the expression of a recombinant protein. The host cell can also be contacted with a composition comprising a concentration, for example a sublethal concentration, of a first compound to which the host cell is sensitive, wherein the first compound causes a decrease in the expression of cellular protein as compared to a host cell that has not been contacted with said first compound.

[0035] Still other embodiments relate to methods of increasing the level soluble recombinant protein relative to the level insoluble recombinant protein expressed in a host cell. These methods can include the steps of: contacting the host cell with a regulator compound, wherein the regulator compound induces the expression of a recombinant protein, and contacting the host cell with a composition comprising a sublethal concentration of a first compound, wherein the first compound causes an increase in the level of soluble recombinant protein as compared to a host cell that has not been contacted with the first compound, wherein the host cell is sensitive to the first compound.

[0036] Other embodiments relate to methods of increasing the ratio of expressed recombinant protein to expressed host cell protein. These methods can include the steps of: providing a host cell that can include endogenous DNA encoding host cell protein and a recombinant nucleic acid construct encoding a recombinant protein, wherein the host cell is sensitive to a first compound; and contacting the host cell with the first compound, wherein the first compound reduces host cell macromolecular synthesis, and wherein the ratio of expressed recombinant protein to expressed host cell protein in the host cell increases after contacting the host cell with the first compound.

[0037] Still other embodiments relate to an improved method for producing a recombinant protein from a recombinant nucleic acid construct in a host cell. The method can include reducing the level of background protein expression in the host cell by administering to the host cell a concentration, for example a sublethal concentration, of a first compound prior to harvesting the recombinant protein, wherein the first compound reduces host macromolecular synthesis.

[0038] Other embodiments relate to a method for producing a recombinant protein. A host cell having a recombinant nucleic acid construct, wherein said recombinant nucleic acid construct operably encodes said recombinant protein can be provided. A sublethal concentration of a first compound that reduces synthesis of native macromolecules by the host cell can be administered to the host cell, and thereafter the recombinant protein can be harvested from the host cell.

[0039] Still other embodiments relate to a method for increasing the ratio of expressed recombinant protein to expressed host cell protein. A host cell can be provided that comprises endogenous DNA encoding host cell protein and a recombinant nucleic acid construct encoding a recombinant protein. The host cell can be sensitive to a first compound. The recombinant nucleic acid construct can render the host cell insensitive to a second compound. The host cell can be contacted with the first compound and the second compound. The first compound can reduce host cell macromolecular synthesis. The ratio of expressed recombinant protein to expressed host cell protein in the host cell can increase after contacting the host cell with an amount, for example a sublethal concentration, of the first compound.

[0040] Other embodiments relate to methods for producing a recombinant protein. A population of cells can be provided that include one or more cells that are susceptible to a first compound. One or more host cells can have a recombinant nucleic acid construct that confers resistance against the second compound, and that operably encodes a recombinant protein. The population of cells can be contacted with the first compound, thereby selecting the host cells having a recombinant nucleic acid construct that have been contacted with the first compound. A sublethal concentration of a second compound can be administered to the host cells having the recombinant nucleic acid construct, wherein the second compound can. . reduce the production of native protein in the host cells. The recombinant protein thereafter can be harvested from the host cells.

[0041] Still other embodiments relate to a method for producing a recombinant protein, in an E, coli host cell with an inducible T7 RNA polymerase system. The host cell can include a recombinant nucleic acid construct, having a T7 promoter operably linked to a nucleic acid encoding the recombinant protein. A concentration, for example sublethal concentration, of an antibiotic or antibiotic analog that reduces the production of native protein by the E. coli host cell can be administered to the host cell. The E. coli host cell thereafter can be harvested and recombinant protein can be obtained from said E. coli host cell.

[0042] Still other embodiments relate to a method of producing a soluble recombinant protein in a host cell that is sensitive to a second compound. The host cell can be contacted with a first compound that increases expression of the recombinant protein. The host cell also can be contacted with a concentration, for example a sublethal concentration of a second compound, wherein the sublethal concentration of the second compound causes an increase in the level of soluble recombinant protein within the host cell.

[0043] Yet other embodiments relate to methods of producing a soluble recombinant protein within an E. coli host cell. An E. coli host cell that has a recombinant nucleic acid construct encoding a recombinant protein can be provided. The host cell can be sensitive to a first compound, wherein the first compound is chloramphenicol, spectinomycin, erythromycin, or kanamycin. The host cell can be contacted with a first composition that induces recombinant protein expression, and a second composition, wherein the second composition includes the first compound and at least one of the following compounds: an alkali salt, an oxidizing agent or an antioxidant, or a reducing agent. The soluble recombinant protein can be obtained from the host cell. [0044] In some embodiments of the above method, the first composition and the second composition can be administered together. For example, the first composition and the second composition can be mixed together prior to administration.

[0045] In some embodiments of the above method, the host cell can be E. coli BL21.

[0046] In yet other embodiments of the above method, the host cell can be contacted with a concentration of about 0.1 to about 35 micrograms per ml of the first compound.

[0047] Other embodiments relate to kits for increasing the levels of a recombinant protein expression relative to cellular protein in a host cell. The kits can include a first compound that increases the ratio of recombinant protein to native host cell protein, wherein the host cell is sensitive to the first compound. In further embodiments, the kit can include a host cell. In some embodiments, a competent host cell can be provided in the kits.

[0048] Other embodiments relate to kits for increasing the solubility of a recombinant protein expressed in a host cell engineered to express the recombinant protein. The kits can include a composition that includes a first compound, wherein the host cell is sensitive to the first compound, and wherein administering a sublethal concentration of the first compound to a host cell that is expressing the recombinant protein increases the level of soluble recombinant protein within the cell.

[0049] In further embodiments of the kits provided herein, the recombinant protein can have a tag. For example, the recombinant protein can have a six-histidine tag. In other embodiments, the kits can include an expression vector for the expression of the recombinant protein. In further embodiments, the expression vector renders the host cell resistant to a second compound. In yet other embodiments, the kits can include a host cell. In further embodiments, the host cell can be competent. For example, in some embodiments, the kits can include competent Escherichia coli BL21(DE3).

[0050] In still other embodiments of the kits described herein, the first compound can be an antibiotic, for example, streptomycin, gentamycin, amikacin, kanamycin, neomycin, netilmycin, paromycin, spectinomycin, tobramycin, erythromycin, azithromycin, clarithromycin, roxithromycin, telithromycin, chloramphenicol, and the like. In some embodiments, the first compound of the kits described herein can exclude one or more of the above-mentioned first compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 shows Western blots comparing the amount of VVl-A9(His₆) in the soluble fraction in E. coli BL21(DE3) overexpressing this protein, in cultures that were untreated or treated with sublethal concentrations of spectinomycin, as described in Example 2.

[0052] Figure 2 shows Western blots comparing the amount of Wl-A9(His₆) in the soluble fraction in E, coli BL21(DE3) overexpressing this protein, in cultures that were untreated or treated with sublethal concentrations of chloramphenicol as described in Example 3.

[0053] Figure 3 shows Western blots comparing the amount of VVl-A9(Hisδ) in the soluble fraction in E. coli BL21(DE3) overexpressing this protein, in cultures that were untreated or treated with sublethal concentrations of erythromycin as described in Example 4.

[0054] Figure 4 shows Western blots comparing the amount of Wl-A9(His₆) in the soluble fraction in E. coli BL21(DE3) overexpressing this protein, in cultures that were untreated or treated with sublethal concentrations of kanamycin as described in Example 7.

[0055] Figure 5 A shows a graph showing the concentration of proteins recovered from NiNTA purification of proteins from E. coli BL21(DE3) cultures expressing recombinant ketosteroid isomerase His₆ fusion protein. The proteins were recovered from the soluble fraction of E. coli BL21(DE3) cultures grown in media with the indicated amounts of spectinomycin. Spectinomycin was added either at the time of induction of recombinant protein expression, or stepwise over time after induction of recombinant protein expression, as described in Example S.

[0056] Figure 5B shows a Colloidal Blue stained get of proteins recovered from NiNTA purification of proteins from E. coli BL21(DE3) cultures expressing recombinant ketosteroid isomerase His₆ fusion protein. The proteins were recovered from the soluble fraction of E. coli BL21(DE3) cultures grown in media with the indicated amounts of spectinomycin. Spectinomycin was added either at the time of induction of recombinant protein expression, or stepwise over time after induction of recombinant protein expression, as described in Example 8.

[0057] Figure 6 shows a series of Western Blots detecting recombinant VVl_A8(His₆) in the soluble fraction (Figure 6A) or insoluble fraction (Figure 6B) of E. coli BL21(DE3) following addition of a sublethal amount of erythromycin, as described in Example 5.

[0058] Figure 7 shows a series of Western Blots detecting recombinant VVl_A8(His₆) in the soluble fraction of E. coli BL21(DE3) cultured in media with elevated levels of NaCl, following treatment with a sublethal amount of erythromycin, as described in Example 6.

[0059] Figure 8 A shows a series of Western Blots detecting total recombinant TB C5 (Hi_S6) in E. coli BL21(DE3) at various timepoints following addition of a sublethal concentration of spectinomycin, as described in Example 10.

[0060] Figure 8B shows a series of Colloidal Blue stained gels, parallel to the Western Blots in Figure 8A, detecting total whole cell proteins in E. coli BL21(DE3), as described in Example 11.

[0061] Figure 8C shows a graph of the cell culture density (A OD₆₀₀) at indicated timepoints after the addition of a sublethal concentration of spectinomycin, as described in Example 11.

[0062] Figure 9A shows a series of Western Blots detecting total recombinant XWI l(Hi_S6) in E. coli BL21(DE3) at various timepoints following addition of a sublethal concentration of spectinomycin, as described in Example 12.

[0063] Figure 9B shows a series of Colloidal Blue stained gels, parallel to the Western Blots in Figure 9A, detecting total whole cell proteins in E. coli BL21(DE3) , as described in Example 12.

[0064] Figure 9C shows a graph of the cell culture density (A OD₆oo) at indicated timepoints after the addition of a sublethal concentration of spectinomycin, as described in Example 12. [0065] Figure 1OA shows a series of Western Blots detecting total recombinant Green Fluorescent Protein in E. coli BL21(DE3) at various timepoints following addition of a sublethal concentration of spectinomycin, as described in Example 13.

[0066] Figure 1OB shows a series of Colloidal Blue stained gels, parallel to the Western Blots in Figure 1OA, detecting total whole cell proteins in E. coli BL21(DE3), as described in Example 13.

[0067] Figure 1 IA shows a series of Western Blots detecting total recombinant Green Fluorescent Protein in E. coli BL21(DE3)ρLysE or in E. coli BL21(DE3)ρLysS as indicated following treatment with a sublethal concentration of erythromycin, as described in Example 13.

[0068] Figure 1 IB shows a series of Colloidal Blue stained gels, parallel to the Western Blots in Figure 11A, detecting total whole cell proteins in E. coli BL21(DE3)pLysE or in E. coli BL21(DE3)pLysS, as indicated, as described in Example 13.

[0069] Figure 12A shows a series of Western Blots detecting total recombinant WlA_9(His₆) in E. coli BL21(DE3) at various timepoints following addition of a sublethal concentration of spectinomycin, as described in Example 14.

[0070] Figure 12B shows a series of Colloidal Blue stained gels, parallel to the Western Blots in Figure 12 A, detecting total whole cell proteins in E. coli BL21(DE3), as described in Example 14.

[0071] Figure 13 A shows a series of Western Blots detecting recombinant VVlA9(His₆) in E. coli BL21(DE3) at various timepoints following addition of at various sublethal concentrations of streptomycin and chloramphenicol, as described in Example 15.

[0072] Figure 13B shows a series of Colloidal Blue stained gels, parallel to the Western Blots in Figure 13 A, detecting total whole cell proteins in E. coli BL21(DE3), as described in Example 15.

[0073] Figure 14 shows a series of Western blots detecting recombinant KSI/Luciferase(His_δ) in E, coli BL21(DE3) host cells cultured with or without spectinomycin and/or erythromycin. "Purified Protein" shows recombinant protein purified under non- denaturing conditions, representing soluble recombinant protein. "Whole Lysate" shows the recombinant protein present in whole cell lysates of the same cultures shown in the "Purified Protein" blot. "Luciferase Assay" shows the luciferase activity of assays performed on whole cell lysates of the cultures shown in the "Whole Lysate" blot. The Experiments are described in Example 9.

[0074] Figure 15 shows a series of Colloidal Blue stained gels showing proteins present in the soluble fraction or whole cell lysates of E. coli BL21(DE3) engineered to express a KSI/Luciferase(His₆) fusion protein, and cultured with or without spectinomycin and/or erythromycin. "Luciferase Assay" shows the luciferase activity of assays performed on whole cell lysates of the cultures shown in the "Whole Lysate" gel. The Experiments are described in Example 9.

[0075] Figure 16 shows a Colloidal Blue stained gel showing proteins present in the soluble fraction or insoluble fraction of cell lysates of E. coli DH5α engineered to express the MMBl protein, and cultured with or without spectinomycin or erythromycin. The Experiments are described in Example 16.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0076] Disclosed herein are improvements in recombinant protein expression and purification. Generally, at least some embodiments relate to methods, systems, and kits for producing recombinant protein. For example, the methods, systems and kits can be used with prokaryotic, eukaryotic, or in vitro expression systems. The methods, systems, and kits can include the use of a compound to which the cell or system is sensitive, for example, a compound to which the cell lacks resistance. For example the compound can be an antibiotic to which the recombinant host cell is sensitive or not resistant. The compound can be contacted with the recombinant host cell/expression system and the recombinant protein can be obtained. The methods, systems and kits can be utilized, for example, to produce soluble recombinant protein, to produce recombinant protein in a greater ratio compared to host cell protein, and to produce recombinant protein with greater specific activity.

[0077] Increasing the ratio of recombinant protein to host cell protein: As mentioned above, some embodiments relate to producing recombinant protein in a greater ratio compared to host cell protein. For example, some aspects relate to methods and kits for enriching the amount of recombinant protein in a host cell relative to endogenous host cell protein, thereby simplifying subsequent purification of the recombinant protein from the multitude of other proteins present in the host cell, referred to herein as "endogenous host cell proteins," "host cell protein," "native protein," or "background protein." For example, some embodiments relate to methods of reducing the levels of background protein, (e.g., all proteins other than the recombinant protein), which renders downstream purification faster and easier, and which can lessen the need for tagging proteins, which may lead to undesirable effects on protein activity. It should be noted that in some aspects proteins can still be tagged. The methods described herein can be combined with any other methods in the art to enhance and improve recombinant protein expression and purification, including in vivo and in vitro expression, including for example to facilitate purification of tagged proteins.

[0078] For example, some embodiments relate to methods of increasing the ratio of expressed recombinant protein to expressed host cell protein. An increase in the ratio of expressed recombinant protein to expressed host cell protein can be achieved, for example, by causing an increase in recombinant protein that is greater than any increase in host cell protein, or by causing a decrease in host cell protein that is greater than any decrease in recombinant protein, or both.

[0079] One example of a method for increasing the ratio of recombinant protein to host cell protein can include the steps of providing a host cell, where the host cell includes endogenous DNA encoding host cell protein and a recombinant nucleic acid construct encoding a recombinant protein, wherein the host cell comprises a selectable marker, and wherein the host cell is sensitive to a first compound, and contacting the host cell with a composition comprising the first compound, wherein the first compound reduces host cell macromolecular synthesis, and wherein the ratio of expressed recombinant protein to expressed host cell protein in the host cell increases after contacting the host cell with the first compound. Preferably, the first compound reduces the concentration of host cell protein. Contacting the host cell with the first compound may reduce the quantity of host cell protein synthesized without significantly reducing the quantity of the recombinant protein. In other preferred embodiments, following the contacting step, the amount of soluble recombinant protein expressed in host cells is greater than the amount of soluble recombinant protein expressed in host cells that have not been contacted with the composition. [0080] Producing soluble recombinant protein; Other embodiments relate to methods and kits for producing soluble protein and for increasing the level of soluble recombinant protein expression within a host cell. Accordingly, the methods and kits disclosed herein facilitate the production and purification of biologically active proteins useful for subsequent research, diagnostic, or therapeutic purposes. Thus, the present methods and kits are useful for the production of recombinant proteins that form insoluble aggregates and/or have low specific activity due to aggregation. For example, some embodiments relate to methods for producing and increasing the level of soluble recombinant protein, thereby avoiding the cost, time, and expense of, altering the recombinant protein itself, (e.g., to harbor a leader sequence), subjecting insoluble protein aggregates to a harsh denaturation/renaturation process, or completely changing host organism. The methods and kits disclosed herein can be combined with any of the methods in the art to enhance solubility of recombinant proteins. For example, the methods can be used with an E. coli host cell genetically engineered to over-express chaperone proteins to increase the level of soluble recombinant protein. Recombinant proteins that have proven difficult to produce in an active form using other methods can be recombinantly expressed using the methods and kits described herein, thereby increasing the recovery of active protein.

[0081] Generally, soluble proteins are present in solution within the cytosol or periplasm of host cells, whereas insoluble proteins are present in aggregates, such as for example in inclusion bodies. The soluble fraction of the host cell can be separated from the insoluble fraction of the host cell using any technique, such as techniques described in Sambrook et al, (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1989]) and/or Ausubel et al, eds, (Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, NY [1994]); each of which is herein incorporated by reference in its entirety. These methods enable the comparison of the amounts or levels of proteins, (e.g., a recombinant protein), in the soluble fraction to the insoluble fraction of the host cells, and between host cells that have been treated with compositions according to the methods described herein, and host cells that have not been treated with the compositions. [0082] As used herein, an "increase" in the level of soluble recombinant protein can refer to an increase in the amount of soluble recombinant protein over a baseline level of soluble recombinant protein found in the soluble fraction of host cells which have not been propagated according to the methods described herein. For example, an increase in soluble protein can arise when there is detectable recombinant protein in the soluble fraction of a host cell in which there is no detectable recombinant protein in the soluble fraction when the host cell is not propagated according to the methods described herein. The increase in the amount of soluble recombinant protein may be accompanied by a decrease in the amount of recombinant protein found in the insoluble fraction of the host cells. The increase in the amount of soluble recombinant protein may not be accompanied by an increase in the total amount of recombinant protein synthesized. The increase in the amount of soluble protein can be accompanied by an increase in the specific activity of the protein.

[0083] The methods and kits disclosed herein can be useful in addressing the problem of limited or decreased activity of recombinant proteins that form insoluble aggregates in in vitro and in vivo recombinant expression systems, that exhibit limited or decreased specific activity. Specific activity is defined as units of activity per units of protein. A recombinant protein with limited or decreased activity is a recombinant protein that, when produced in an in vivo or an in vitro system has a lower specific activity when compared to counterpart proteins that have not been recombinantly expressed. Accordingly, a recombinant protein that has limited or decreased activity may be identified, and produced by the methods disclosed herein. Producing the recombinant proteins by the methods disclosed herein may yield recombinant protein having a specific activity higher than recombinant protein that has not been produced by the methods disclosed herein.

[0084] The methods, compositions and kits disclosed herein can be used to increase the solubility of numerous recombinantly-expressed proteins. Examples include G- protein coupled receptors, or GPCRs, protein kinases, and other proteins. Non-limiting examples of GPCRs include monoamine receptors such as serotonin-binding, adrenaline- binding, dopamine-binding, histamine-binding, and acetylcholine-binding receptors. Other classes of GPCRs include protease activated receptors (PARs) and metabotropic glutamate receptors (mGluRs). Non-limiting examples of protein kinases include the receptor tyrosine kinases (RTK's), including but not limited to the following families of proteins: EGFR, INSR, PDGFR, FGFR, VEGFR₅ EPH, TRK, AXL₅ TIE, MET₅ DDR, RET₅ ROS, ALK₅ ROR₅ RYK, PTK7 and MUSK. The methods, compositions and kits can also be used in the production of other proteins such as mTOR (GENBANK™ accession No. NP_004949), herein expressly incorporated by reference in its entirety.

[0085] As mentioned above, some embodiments relate to methods of producing a recombinant protein. The methods can include the step of contacting a host cell or in vitro expression system that is sensitive to a first compound with the first compound to which it is sensitive, and which host cell or in vitro expression system includes a recombinant nucleic acid construct encoding a protein. Other embodiments relate to methods of contacting an in vitro system with such first compounds. The various methods can further include the step of obtaining the expressed protein from the host cell or from the in vitro system. The methods can include additional or different steps, which are further described herein.

[0086] The term "recombinant protein" refers to a protein that is produced using molecular biology techniques, for example, recombinant DNA technology. As an example, "recombinant protein" can refer to a protein from a genetically engineered nucleic acid, such as a "recombinant nucleic acid construct." Any protein, peptide, or polypeptide can be encoded by an engineered nucleic acid construct or recombinant nucleic acid construct. The term "protein expression" refers to the processes of transcription and translation of nucleic acids to produce polypeptides.

[0087] As used herein, the term "host cell" can refer to a cell or a population of cells harboring or capable of harboring a recombinant nucleic acid construct. Preferably, the recombinant nucleic acid construct can be compatible with the host cell, for example, capable of autonomous replication in the host cell or stably integrated into one of the host cell's chromosome(s). Further, a suitable host cell can have the requisite cellular machinery to transcribe and translate the recombinant mRNA and protein, provided that the recombinant nucleic acid construct contains the proper replication, transcription, and translation signals correctly arranged. Further, in aspects where the nucleic acid encoding the recombinant protein is operably linked to a promoter recognized by a heterologous RNA polymerase, the host cell can comprise a heterologous RNA polymerase that recognizes and transcribes the construct that encodes the recombinant protein.

[0088] Preferably the host cell can be a prokaryotic cell. As used herein, the term "cell" can mean a single cell or a plurality of cells. Several features render prokaryotes particularly suitable as hosts for the expression of recombinant proteins. Prokaryotic cells can be grown to high densities and have minimal nutrient requirements, and are often well characterized. Any prokaryotic cell can be used. Preferably, in some aspects Gram negative or Gram positive cells can be used. Without being limited thereto, an exemplary Gram negative cell is Escherichia coli (E. coif). Non-limiting examples of E. coli strains suitable for use as host cells, include BL21, BL21(DΕ3), DH5α, JM109, JMlOl, HBlOl, BL21(DE3) pLysS, BL21(DE3) pLysE, DHlOB, XLl-Blue, XLlO-GoId, NM522, TOPlO, and the like. Other exemplary Gram negative bacteria useful in the methods described herein include bacterial of the genus Salmonella or Pseudomonas. Gram positive bacteria, such as for example members of the genus Bacillus, including Bacillus subtilis are also useful in the embodiments described herein. Several different strains of B. subtilis are amenable for recombinant protein expression, including but not limited to MC4100, JH642, and the like.

[0089] Alternatively, the host cells can be eukaryotic, for example, yeast cells, insect cells, and various animal cells, such as mammalian cells. Yeast cells provide many of the same advantages as prokaryotic cells for recombinant protein expression including the ability to grow the cells to high cell densities and the minimal nutrient requirements. As an example, preferably, yeast cells belonging to the family Saccharomycetes, such as Saccharomyces ceήvisiae and Pichia pastoris can be used as host cells. As an additional example, yeast cells belonging to the family Schizosaccharomycetes, such as Schizosaccharomyces pombe can be used as host cells. Exemplary mammalian cells, include but are not limited to, CHO cells, COS- 7 cells, 293 cells, NIH-3T3 cells and the like. Additionally, insect cells such as Sf-9 cells, and the like can be suitable host cells.

[0090] As mentioned above, the host cells can be sensitive to a first compound. As used herein in some embodiments, the term "sensitive" can mean that the host cell is not naturally resistant to, or is not genetically engineered to be resistant (i.e., by harboring a recombinant nucleic acid construct encoding a gene whose product confers resistance to the compound) to a compound that causes the host cells to cease propagating, (i.e., the cells are killed), when the cells are contacted with an amount of the compound.

[0091] The host cells and in vitro systems can be contacted with the first compound, In the case of a host cell system, the cell can be contacted with an amount of the first compound, for example, a lethal or sublethal amount. A "sublethal amount" refers to an amount of a compound to which a cell is sensitive that does not result in a complete cessation of propagation of the cell upon contacting the cell with the compound. Accordingly, cultures of cells contacted with a sublethal amount of the compound can remain capable of resuming growth. In other aspects, the host cell can be contacted with an amount of the first compound that ultimately is lethal to the host cell.

[0092] By way of example, the amount of the compound administered to or contacted with the host cell can be about 0.01 μg/ml to about 75 μg/ml. For example, the concentration of the compound can be at least 0.01, 0.1, 0.2, 0.5, 1.0, 2.0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 μg/ml. Preferably, the amount of the compound can be between 0.1 to 60 μg/ml. Preferably, the amount of the compound can be between 0.5 to 50 μg/ml. In most preferred embodiments, the amount of the compound can be 1.0 to 35 μg/ml.

[0093] Following contact with the first compound, macromolecular synthesis within the host cell can be reduced and the level of recombinant protein expressed can be increased relative to endogenous host proteins. As used herein, "macromolecular synthesis" can refer to the synthesis of DNA, RNA and protein. For example, some aspects relate specifically to reducing the synthesis of host cell protein (i.e., endogenous or native proteins not encoded by a recombinant nucleic acid construct). The reduction in host cell protein synthesis does not necessarily accompany a reduction in the quantity of recombinant protein synthesized.

[0094] As used herein, the term "first compound" can refer to any compound to which the host cell is sensitive. As mentioned above, in some aspects the first compound can be a compound that reduces host cell macromolecular synthesis. Generally, the first compound can be any substance that kills or inhibits the growth or viability of a host cell, such as a bacterium, a yeast, a mammalian, or an insect cell. Also, the first compound can include any compound that inhibits an in vitro expression system.

[0095] In sonie embodiments the first compound can be used alone, while in other embodiments the first compound can be part of a composition or cocktail that can include additional components such as buffers, salts, solvents, osmoprotectants, oxidizing agents or antioxidants, reducing agents, and the like. In some preferred embodiments, at a minimum, the methods and kits can include the first compound, but are not limited to only the first compound. Furthermore, in some embodiments, one or more additional compounds can be used in the described methods and kits, preferably one or more that reduce host cell macromolecular synthesis. For example, a cocktail can be used that includes the first compound, as well as one or more additional compounds either that reduce macromolecular synthesis, (e.g., a second, third, fourth compound, etc.), or that aid host cell growth and survival when challenged with one or more other compounds.

[0096] In embodiments where the host cell is prokaryotic, preferably, the first compound can be an antibiotic or an analog of an antibiotic. Several antibiotics work by targeting the synthetic machinery of the cells, such as molecules responsible for DNA, RNA or protein synthesis, and thereby affect macromolecular synthesis in cells that are sensitive to the compound. "Analogs" of antibiotics refer to non-naturally occurring compounds that have similar chemical properties to other antibiotics. Some analogs can have improved antibiotic activity, while others may have a similar activity to the parent antibiotic, while others can have a different type of antibiotic activity. Preferably, the antibiotics and analogs in the present embodiments interfere with host cell protein synthesis. Any antibiotic or analog is contemplated for use with the methods described herein.

[0097] Several antibiotics affect translation of mRN A into protein. Ribosomes are multisubunit ribonucleotprotiens the molecules that translate mRNA into protein. Ribosomes consisting of two subunits: the small ribosomal subunit and the large ribosomal subunit. . In prokaryotes, ribosomes have a 30S and a 50S subunit. In some aspects, the first compound can target the 30S subunit of prokaryotic ribosomes and thereby reduce protein synthesis in the host cell. For example, the first compound can be an aminoglycoside, such as streptomycin, gentamycin, amikacin, kanamycin, neomycin, netilmycin, paromycin, spectinomycin, or tobramycin, or the like. In other aspects of the invention, the first compound can target the 5OS subunit of prokaryotic ribosomes and thereby affect prokaryotic protein synthesis. Non-limiting examples of first compounds that target the 5OS subunit of ribosomes are macrolides, lincosamides, lincosides, puromycin or chloramphenicol. Compounds such as erythromycin, azithromycin, clarithromycin, roxithromycin, telithromycin and the like are non-limiting examples of macrolides that are useful in aspects of described embodiments.

[0098] Some compounds affect protein synthesis in both prokaryotic and eukaryotic host cells, such as puromycin and the like, and can be used in embodiments where the host cells are either prokaryotic or eukaryotic, provided, of course, that the cells are sensitive to the compound. Other compounds affect macromolecular synthesis in primarily in eukaryotic cells, such as, cycloheximide, hygromycin, G-418, diphtheria toxin, blasticin S, and doxyrubicin, and can be suitable in embodiments wherein the host cell is eukaryotic. Any other compound can be used with eukaryotic cells.

[0099] Those skilled in the art are familiar with the compounds disclosed above, as well as the amounts of those compounds that are lethal or sublethal to sensitive host cells. The skilled artisan is familiar with the concentrations of compounds such as antibiotics that are frequently used to prevent growth or kill cells, and therefore can readily determine a range of sublethal concentrations of compounds. The methods described in Examples 20 to 22 describe how such concentrations are readily ascertainable using methods known to those skilled in the art.

[0100] Exposure of bacterial cells to conditions of high osmolarity (e.g., IM NaCl) causes the cells to undergo a stress response, resulting in global changes in macromolecular synthesis, which are not fully understood. Bacterial and eukaryotic cells control cytoplasmic osmolarity by accumulating osmolytes. See, Barth et al., J. Appl. Env. Microb., (2000), 66(4): 1572-1579, herein incorporated by reference in its entirety. In embodiments described above and elsewhere, methods further involve contacting the host cell with a salt, in addition to the first compound. Thus, in some embodiments, prokaryotic host cells can be contacted with an alkaline metal salt, for example sodium chloride (NaCl), in addition to a first compound. In further aspects, the prokaryotic host cells can be contacted with between about 1 mM and IM NaCl. In preferred embodiments, prokaryotic host cells can be contacted with between about 100 mM and IM NaCl, preferably between about 300 mM and 600 mM NaCl, and most preferably with about 400 mM NaCl.

[0101] In still further embodiments, prokaryotic host cells can be contacted with an osmoprotectant. By way of example, the host cells may be contacted with osmoprotectants such as glycine, glycinebetaine, ectoine, sugars, carnitine, proline, glutamate, sorbitol, and mannitol, and the like. Concentrations of both salts and osmoprotectants that are tolerated by prokaryotic host cells are known to those skilled in the art. For example, in some embodiments, host cells can be contacted with 1OmM glycinebetaine.

[0102] Additionally, the host cells may be contacted with an oxidizing agent or an antioxidant (such as hydrogen peroxide) or a reducing agent (for example, dithiothreitol, 2- mercaptoethanol). For example, the prokaryotic host cells can be contacted with 0.1% hydrogen peroxide.

[0103] Thus, in some aspects a recombinant E. coli host cell, such as BL21 (DE3), that is sensitive to spectinomycin can be contacted with approximately about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 μg/ml spectinomycin. In other aspects, a recombinant E. coli host cell, such as BL21(DE3), that is sensitive to chloramphenicol can be contacted with, for example, approximately about 0,2, 0.5, 1.0, or 2.0 μg/ml chloramphenicol. In still other aspects, a recombinant E. coli host cell, such as BL21(DE3), that is sensitive to kanamycin can be contacted with, for example, approximately about 0.2, 0.5, 1.0, or 2.0 μg/ml kanamycin. In other aspects, a recombinant E. coli host cell, such as BL21(DE3), that is sensitive to erythromycin can be contacted with, for example, approximately about 5, 10, 20, 35 or 50 μg/ml erythromycin. .

[0104] As mentioned above, some embodiments include a recombinant nucleic acid construct or an expression vector. In some aspects, the recombinant nucleic acid construct or expression vector encoding the recombinant protein can include a selectable marker that renders the host cell resistant to a selection compound, which would kill or cause the host cell to stop proliferating in the absence of the selectable marker. Some embodiments, including those described above and elsewhere herein, can include a selecting step. For example, some embodiments can include the steps of providing a population of cells, wherein the population includes one or more cells that are susceptible to a selection compound and one or more host cells having a recombinant nucleic acid construct, wherein the recombinant nucleic acid construct encodes a selectable marker that confers resistance against the selection compound, and wherein the recombinant nucleic acid construct operably encodes a recombinant protein; contacting the population of cells with the selection compound; selecting one or more host cells having a recombinant nucleic acid construct that have been contacted with the selection compound; and administering a sublethal concentration of a first compound to the selected one or more host cells having a recombinant nucleic acid construct, wherein the first compound reduces macromolecular synthesis in the host cells, for example, by slowing translation in the host cells; and thereafter harvesting the recombinant protein from the host cells. Contacting the host cells with the first compound may also result in an increase in the level of recombinant protein in the soluble fraction of the host cells.

[0105] Selectable markers useful in both prokaryotic and eukaryotic host cells are well-known in the art. For example, see, Ausubel, et ah, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (2004), and SIGMA-Aldrich Catalog; SIGMA Aldrich (2004): St. Louis, MO; for a comprehensive list of commonly used antibiotics for bacterial, plant, and mammalian cell culture, each of which is herein incorporated by reference in its entirety. Alternatively, complementation can be used as a mechanism to select for host cells that harbor the recombinant nucleic acid construct, wherein host cells that lack a functional copy of a gene required for growth in certain conditions ("selection conditions") are provided with a recombinant nucleic acid construct with a functional copy of the gene. The host cells can be grown under selection conditions, and those host cells harboring the recombinant nucleic acid construct will continue to grow, whereas the cells lacking the recombinant nucleic acid construct will not grow.

[0106] Growth conditions, such as the culturing media and temperature for host cells, also can affect intracellular processes such as macromolecular synthesis and protein folding. As such, growth conditions can affect the levels of recombinant protein compared to endogenous host cell protein, and/or the level of soluble recombinant protein compared to insoluble recombinant protein. Growth and culture conditions for typical host cells are well- known to those of skill in the art. For example, growth and culturing conditions can be carried out as described in Ausubel, et al., supra, Cereghino JL, Cregg JM, Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. (2000) Jan;24(l):45-66, and O'Reilly et al. (1992), Baculovirus Expression Vectors: A Laboratory Manual (Oxford Univ. Press, New York); each of which is herein incorporated by reference in its entirety.

[0107] Some embodiments can include culturing the host cells at a particular temperature or temperature range. Many preferred temperatures and ranges are well known to those of skill in the art. For example, preferably the prokaryotic cells can be cultured between about 4 C to 42 C. Optionally, prokaryotic host cells can be cultured between about 16^°C and 42^°C, or between about 30^°C and 37^°C. Most preferably, the prokaryotic host cells can be cultured at approximately 30 C. Yeast host cells can preferably be cultured between about 4^°C to 37^°C. and most preferably at 24^°C. Mammalian and insect host cells can preferably be cultured between 20^°C to 42^°, and most preferably at 37^°C.

[0108] Other embodiments, including those described above and elsewhere herein can include a regulation step, for example, an induction step. For example, expression of the recombinant protein can be regulated by a regulator compound, such as an inducer compound that induces recombinant protein expression. Transcription from some promoters can be regulated by adding one or more regulator compounds to the culture. For clarity, for the purposes of this description, a compound that regulates expression of the recombinant protein is referred to as a "regulator compound." In some aspects, the regulator compound can be an "induction" or "inducer" compound, and can result in an increase in the expression of recombinant protein-following contacting the host cell with the regulator compound.

[0109] As used herein, the term "inducer" refers to a compound that functions either directly or indirectly, for example by functioning as an anti-repressor, to increase the expression of an inducible gene. Recombinant protein expression in host cells can also be regulated, including being induced, by changing the growth media, for example from one carbon source to another. Pichia pastoris is an example of a type of cell that can be regulated by providing different carbon sources to regulate the growth phase and the induction phase for producing the recombinant protein. Other regulation systems can be used. Many are known by those of skill in the art.

[0110] Inducible expression systems for recombinant protein expression in eukaryotic systems are well known to those of skill in the art. Examples of vectors that allow inducible expression of recombinant proteins in yeast such as Saccharomyces cerivisiae include pYepSecl (Baldari, et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Similar expression vectors are known for Pichia, such as pFLD (Invitrogen Corp., San Diego, Calif.), and the like. Examples of vectors for inducible recombinant protein expression in mammalian cells include pMex-NeoI, pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Examples of vectors for inducible recombinant protein expression in insect cells include baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) MoI. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V. A. and Summers, M. D. (1989) Virology 170:31-39). For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0111] In some embodiments, the recombinant nucleic acid construct or expression vector can include a heterologous promoter operably linked to the nucleic acid encoding the recombinant protein. As used herein, the term "heterologous promoter" refers to a promoter sequence other than the naturally occurring promoter sequence for a nucleic acid and drives transcription of the nucleic acids to which it is "operably linked." Heterologous promoters can be promoters that are not naturally operably linked to the nucleic acid of interest, for example, a promoter form a different genetic entity. When a nucleic acid is "operably linked," it can be placed in a functional relationship with another nucleic acid. [0112] The host cell can comprise a heterologous RNA polymerase, which can be an RNA polymerase from a different genetic entity. Preferably, the heterologous promoters can contain genetic regulatory elements such as enhancers, operators, and the like so that transcription therefrom can be easily manipulated by adjusting the culturing conditions. The gene encoding the heterologous RNA polymerase can be integrated into the host cell chromosomal DNA. Alternatively, the gene encoding the heterologous RNA polymerase can be provided on DNA that is not integrated into the chromosome, for example on plasmid DNA, viral DNA, or any other means to introduce heterologous DNA into host cells.

[0113] Various heterologous promoters can be suitable for use where the host cell is a prokaryotic host cell. Examples of inducible heterologous promoters used in prokaryotes include bacterial promoters such as the Pi_ac, P_trp, P_tac promoters. Transcription from these promoters can be regulated by supplying the host cells with a regulator compound. For example, transcription from the P|_ac or P_tac promoters increases when the host cells are contacted with Isopropyl-Beta-d-Thiogalactopyranoside (IPTG), thereby inducing transcription of nucleic acids operably linked to these promoters. In some aspects, the host cells can be engineered to have transcription of a bacteriophage RNA polymerase, such as T7, T3, SP6 or λ RNA polymerase, operably linked to an inducible promoter. Such host cells are known in the art, such as for example, the E. coli BL21(DE3), and variants thereof such as BL21(DE3) pLysS BL21(DE3) pLysE and the like. In further aspects, the nucleic acid encoding the recombinant protein can be operably linked to promoters recognized by the heterologous RNA polymerase. In other aspects, the gene encoding the recombinant protein of interest can be operably linked to an inducible promoter recognized by the host cell's native transcription machinery.

[0114] In host cells where expression of a heterologous RNA polymerase is under control of an inducible promoter, the nucleic acid encoding the recombinant protein can be operably linked to a cognate promoter that is recognized by the heterologous RNA polymerase. For example, in host cells engineered to express T7, T3, SP6 or λ RNA polymerase, the recombinant protein can be linked to a Pχ₇, Py₃, P_SP6_> λPL, λPR promoter. In the presence of inducer, the RNA polymerase is expressed, thereby leading to expression of the recombinant protein. [0115] In some embodiments the timing of when the host cells are contacted with the regulator compound can affect the ratio of recombinant protein relative to endogenous host cell protein, or the levels of recombinant protein found in the soluble fraction of the host cells. In preferred embodiments, where expression of the recombinant protein is inducible by a regulator compound, the host cell can be contacted with the first compound and the regulator compound at substantially the same time. Alternatively, the host cell can be contacted with the first compound before or after the host cell has been contacted with the regulator compound. For example, the first compound can be administered to the host cell at approximately 10, 20, 30, 45, 60, 120, 150, 180, 210, 240, 300, 360, or 420 minutes after the inducer is administered to the host cells. Preferably, the host cell is contacted with the first compound from about 30 minutes to about 60 minutes, from about 30 minutes to about 90 minutes, from about 60 minutes to about 90 minutes, from about 60 minutes to about 120 minutes, from about 120 minutes to about 180 minutes, from about 120 minutes to about 210 minutes, from about 180 minutes to about 270 minutes, from about 180 to about 300 minutes, or from about 210 minutes to 420 minutes, after contacting the host cell with the regulator compound, such as an inducer. In some embodiments, the host cell can be contacted with the first compound before the host cell is contacted with a regulator compound. Alternatively, the growth of the host cell culture can be monitored by methods well known to those of skill in the art, and the first compound can be administered to host cells when the cells have reached a predetermined density. Methods of measuring the cell density of prokaryotic cells, such as measuring the amount of light absorbed by samples of the cultures at certain wavelengths, are known to those of skill in the art and are described in Ausubel et al., supra. For example, in embodiments wherein the host cell is a prokaryotic, the first compound can be added when the absorbance units of the culture at 600 nm (OD_OOO) is between .01 and 0.5. In some aspects, the first compound can be added when the optical density is .01, or .05, or 0.1, 0.2, 0.3, 0.4, or 0.5.

[0116] In further embodiments of the methods wherein expression of the recombinant protein is induced by a regulator compound, the first compound (or mixture) may be administered in one dose. In other embodiments, the first compound (or mixture) may be administered at multiple times during the expression phase, thereby increasing the amount of first compound with which the host cell is contacted in steps, rather than at once. For example, in some embodiments, the host cell can be contacted with the regulator compound and a first dose of the first compound, and subsequently contacted with one or more doses of the first compound over time, until the host cell is contacted with the desired concentration of first compound. For example, in some embodiments, the host cell is an E, coli cell that is sensitive to spectinomycin. The E. coli cell is contacted with a regulator compound and with a dose of spectinomycin to give a final concentration of 5μg/ml spectinomycin, at approximately the same time. Alternatively, the first dose of spectinomycin can be administered at approximately 30 minutes or 60 minutes after the regulator compound. Approximately 50 minutes later, the host cell is contacted with another dose of spectinomycin, to give a final concentration of lOμg/ml spectinomycin. Approximately every 50 minutes, the host cell is contacted with another dose of spectinomycin until the final concentration of spectinomycin is 35μg/ml spectinomycin. Similarly, compositions that include more than one compound that reduces macromolecular synthesis can be administered to the host cell in several doses, such as a spectinomycin/erythromycin mixture, thereby increasing the concentration of both spectinomycin/erythromycin over time. The host cells are subsequently harvested and the recombinant protein obtained therefrom. In some embodiments, the recombinant nucleic acid can be engineered such that the recombinant protein of interest is "tagged." A non-limiting example of a tag is six consecutive histidine residues that are engineered to be preceded or succeed the amino or carboxy terminus of the protein, respectively. Other protein tags that are well known in the art include the maltose binding protein, glutathione-S-transferase, and the like.

[0117] Preferably the host cell can be maintained following contact with the first compound to permit expression of the recombinant protein. As used herein, the term "maintained" refers to continued host cell viability, growth, and/or continued expression of the recombinant protein. In some aspects, the host cell can be maintained by providing growth media to the host cell.

[0118] In further aspects, the methods can include the step of isolating the recombinant protein after harvesting the host cells. The recombinant protein can be isolated from the harvested host cells using methods known to those skilled in the art, However, because of the methods described herein, the isolation of purification can be improved, for example, because of the increased ratio of recombinant protein to host cell protein,

[0119] In some embodiments where the host cells are prokaryotic, the host cells can be harvested approximately 1 hour after the host cells are contacted with the first compound, while in other embodiments the host cells can be harvested approximately 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 24, or 36 hours after the host cells are contacted with the first compound. In some embodiments the host cells can be harvested between about 1-2 hours after contact with the first compound, about 1-3 hours, about 2-5 hours, about 3-7 hours, or about 5 -10 hours after contact with the first compound, or about 12-24 hours after contact with the first compound.

[0120] It may be desirable to produce the recombinant protein in vitro, for example in a cell free system. Accordingly, as mentioned above, some embodiments include methods of producing soluble protein, including methods of increasing the level of a soluble recombinant protein relative to the level of insoluble protein produced in an in vitro system. The methods systems and kits can be used with any in vitro recombinant expression system.

[0121] Different in vitro systems are available based upon the particular starting genetic material: RNA or DNA. Standard translation systems, such as reticulocyte lysates, wheat germ extracts, and E. colt extracts use RNA as a template; whereas "coupled" and "linked" systems start with DNA templates, which are transcribed into RNA then translated.

[0122] In some embodiments, an in vitro translation system can be utilized. For example, in some aspects, cell-free translation systems such as extracts from rabbit reticulocytes, wheat germ and E, coli are provided. In vitro translation systems can be prepared as crude extracts containing all the mactomolecular components (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNfA. To ensure efficient translation, each extract can be supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (e.g., creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for E. coli systems), and other co- factors such as Mg²⁺, K⁺, and the like. In embodiments that utilize eukaryotic translation systems, the RNA encoding the recombinant protein of interest can contain a 5' cap structure. The mRNA cap is a 7-methyl-guanosine in a 5 -5' linkage with the 5'-terminal nucleotide of the transcribed RNA, and can enhance the efficiency of translation of the recombinant mRNA.

[0123] In embodiments where an in vitro translation system is provided, a recombinant RNA encoding a recombinant protein of interest that is operably linked to translational regulatory elements recognized by the in vitro translation system. The recombinant RNA is combined with the in vitro translation system. An amount of a compound that slows the translation of the recombinant protein is provided. For example, in eukaryotic translation systems, compounds that affect eukaryotic translation machinery, such as cycloheximide, hygromycin, G-418, diphtheria toxin, blasticin S, and doxyrubicin can be added to the in vitro system. On the other hand, in prokaryotic translation systems, compounds that affect prokaryotic translation machinery such as antibiotics or analogs of antibiotics, can be added to the in vitro system. Accordingly, macrolides, aminoglycosides, lincosides, and lincosamides and the like can be added to prokaryotic translation systems.

[0124] In some embodiments, a "coupled" in vitro system is provided. Both eukaryotic and prokaryotic coupled systems are known and available. Non-limiting examples of eukaryotic coupled systems include for example, rabbit reticulolysate systems and Agrobacterium tumefaciens systems {See, e.g., TNT® Coupled transcription translation system, Promega, Madison, WI; Kartasova T, et al, (1981), Nucl. Acid. Res. 9(24):6763-72. Non-limiting examples of prokaryotic coupled systems include E. coli systems and Staphylococcus aureus systems (See, e.g., PROTEINScript™ in vitro transcription and translation system, Ambion, Austin, TX; Murray, R., et al. (2001) Antimicrob. Agents and Chemother., 45(6):1900-1904).

[0125] In embodiments where a coupled in vitro transcription-translation system is provided, a recombinant DNA encoding a recombinant protein of interest that is operably linked to transcriptional and translational regulatory elements recognized by the in vitro translation system. The recombinant DNA is combined with the in vitro coupled transcription translation system. An amount of a compound that slows the translation of the recombinant protein is provided. For example, in eukaryotic coupled transcription translation systems, compounds that affect eukaryotic translation machinery, such as cycloheximide, hygromycin, G-418 and diphtheria toxin, can be added to the in vitro system. On the other hand, in prokaryotic translation systems, compounds that affect prokaryotic translation machinery such as antibiotics or analogs of antibiotics, can be added to the in vitro system. Accordingly, macrolides, aminoglycosides, lincosides, and lincosamides and the like can be added to prokaryotic coupled transcription translation systems.

[0126] In the embodiments wherein recombinant proteins are expressed in vitro, the first compound can be combined with the in vitro system before the recombinant nucleic acid is combined with the in vitro system. Alternatively, the first compound and recombinant nucleic acid can be combined with the in vitro system at approximately the same time.

[0127] Also, the methods described herein can include a further step of producing a crystal of the recombinant protein. Further embodiments relate to methods of producing crystals of proteins. Crystallization methods can be performed according to any method known in the art, including, but not limited to vapor diffusion methods such as hanging drop vapor diffusion and sitting drop vapor diffusion, dialysis methods such as microdialysis, liquid diffusion methods such as capillary counter-diffusion, and high-throughput variations of such methods. Conditions to be used for crystallization trials can include any solutions known for protein crystallization, which can include a precipitant, a salt, a buffer, or one or more additives, and combinations thereof. Exemplary components of a crystallization solution include, but are not limited to polyethylene glycols, methylpentanediol, or salt such as ammonium sulfate or sodium chloride, Exemplary crystallization solutions can be obtained from manufacturers such as Hampton Research (Aliso Viejo, CA). Optimization and modification of crystallization conditions can be performed as is known in the art.

[0128] Also provided are methods of generating antibodies or antibody fragments that specifically bind a recombinant protein. A recombinant protein produced by any of the methods described above can be used to generate an antibody against that recombinant protein, or an epitope on that recombinant protein.

[0129] Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent can include the recombinant polypeptide produced by the methods described herein. It may be useful to conjugate the immunizing agent (e.g., the recombinant protein produced by the methods described herein) to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

[0130] Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103; which is hereby expressly incorporated by reference in its entirety. Mice, such as Balb/c, can be immunized with the recombinant protein produced by the methods described herein. The recombinant protein can be emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen can be emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads. The immunized mice can then be boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice can also be boosted with additional- immunization injections. Serum samples can be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti- PRO antibodies.

[0131] After a suitable antibody titer has been detected, the animals "positive" fox antibodies can be injected with a final intravenous injection of the recombinant protein. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.l, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

[0132] The hybridoma cells can be screened in an ELISA for reactivity against the recombinant protein. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against the recombinant protein is within the skill in the art.

[0133] The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the monoclonal antibodies that specifically bind to the recombinant protein or an epitope on the recombinant protein. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed to purify antibodies.

[0134] Also provided herein are methods of performing biological assays on a recombinant protein. A recombinant protein produced by the methods described herein is provided, arid used in a biological assay. As used herein, the term "biological assay" can refer to any assay that measures an activity or characteristic of the specimen (e.g., a recombinant protein) being tested. By way of example, recombinant proteins produced by the methods described herein can be used in biological assays to detect interaction of the recombinant protein with other proteins, or in assays to identify compounds such as small molecules that interact with the recombinant protein, and possibly modify the activity of the recombinant protein.

[0135] In other embodiments, a kit that increases the solubility of a recombinant protein expressed in a host cell is provided. The kit can include a first compound. Preferably, the host cell is engineered to express the recombinant protein is sensitive to the first compound. The administration of the first compound, for example a sublethal dose of the first compound, can result in an increase in the level of soluble recombinant protein within the host cell. In some embodiments, the kits can also include written instructions, for example instructions providing a protocol for performing the method. [0136] In certain embodiments, the kit can also include a host cell, wherein the host cell is sensitive to the first compound. The host cell can be "competent." As used herein, the term "competent" refers to host cells that are receptive to the uptake of exogenous DNA, such as a recombinant nucleic acid. Examples of suitable host cells are discussed above.

[0137] Preferably, the first compound can be an antibiotic or an analog of an antibiotic. Antibiotics constitute a class of compounds that inhibit or slow growth of microorganisms and viruses. For example, various antibiotics work by targeting the synthetic machinery of the cells, such as molecules responsible for DNA, RNA or protein synthesis, and thereby affect macromolecular synthesis in cells that are sensitive to the compound. "Analogs" of antibiotics refer to non-naturally occurring compounds that have similar chemical structures to other antibiotics. Some analogs can have improved antibiotic activity, while others may have a similar activity to the parent antibiotic, while others can have a different type of antibiotic activity. Preferably, the analog reduces host cell macromolecular synthesis, most preferably, host cell protein synthesis. Any antibiotic or analog is contemplated for use with the methods described herein.

[0138] While particular embodiments of the invention have been described in detail, it will be apparent to those skilled in the art that these embodiments are exemplary rather than limiting. The following are non-limiting examples of some of the embodiments.

EXAMPLES

Example 1 : General Protocol for Producing Soluble Recombinant Proteins Expressed in E. coli

[0139] Recombinant DNA techniques known to the skilled artisan as described in Ausubel et ah, supra, are used to clone the gene encoding the protein of interest into an expression vector that facilitates inducible expression of the recombinant protein. The recombinant expression vector is used to transform an E. coli strain using standard methods.

[0140] Following transformation, the cells are plated on selective media to isolate recombinant host cells. An isolated recombinant colony is used to inoculate 2 ml LB media {See, Ausubel et a!., supra), supplemented with the same concentration of the same antibiotic used to select for recombinant host cells. The culture is grown for approximately 12-16 hours at 30⁰C, shaking. The culture is diluted either 1/5, 1/10/ 1/20, 1/50, 1/100, 1/500, or 1/1000 into LB supplemented with the same antibiotics as the 2 ml culture, a sufficient amount of an inducer to induce expression of the recombinant protein, and appropriate concentration of spectinomycin is added at appropriate time(s). The culture is grown for approximately 2 hrs, 3 hr, 4 hr, 6 hr, 8 hr, 12 hr, or-16 hours at 25⁰C, 30°C, 37⁰C shaking. Cells are harvested by centrifugation, and the recombinant proteins are isolated using standard methods. Example 2: Sublethal Amounts of Spectinomycin Increases the Solubility of a Hisg Tagged

Vaccinia Virus 1 A9 Fusion Protein in E. coli

[0141] The following Examples disclose methods of increasing the solubility of Vaccinia virus A9 (HiS₆) protein in E. coli host cells.

[0142] Vaccinia virus is the causative agent of smallpox in humans, and therefore its proteins are normally expressed in eukaryotic host cells. The solubility of a histidine- tagged version of the VVI- A9 protein over-expressed in E coli cultured with or without the presence of a sublethal amount of antibiotic was tested as described below.

[0143] An expression vector was engineered to overexpress a C-terminal His₆ fusion protein of the Vaccinia virus A9 protein. The polymerase chain reaction (PCR) was used to amplify the gene encoding the Vaccinia virus A9 protein ("VVI-A9") The primers used in the amplification reaction contained Xbal and BamHI restriction enzyme recognition sites to facilitate later cloning. The purified PCR product was digested with Xbal and BamHI and purified using preparative agarose gel electrophoresis and elution. The purified PCR product was cloned into the Xbal and BamHI sites of pET3a (Novagen, Madison, WI), such that six histidine residues ("His₆") were fused in frame to the carboxy terminal end of VVl- A9, to create VVI-A9(His₆). The resulting plasmid was transformed into competent E. coli BL21 (DE3) (Novagen, Madison, WI), according to the manufacturer's instructions. Recombinant host cells were selected by plating the transformation on LB agar plates (See, Ausubel et al, supra), supplemented with 100 μg/ml carbenecillin.

[0144] A carbenecillin resistant colony was isolated and used to inoculate a 2 ml culture of LB medium supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 3O⁰C, shaking. The following morning, the overnight culture was diluted 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, and 200 μg/ml IPTG, to induce expression of the recombinant protein. Parallel cultures were inoculated by diluting the overnight culture 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, 200 μg/ml IPTG, and 1, 2, 5, 10, 20, 35, 50, or 100 μg/ml spectinomycin.

[0145] The cultures were grown for 12-16 hours, at 30⁰C. The cells from 1 ml of each culture were harvested by centrifugation. The cells were resuspended in lOOμl Tris - HCl buffer (pH 8.0), and sonicated using a Branson sonifier 450. Insoluble proteins were isolated from soluble proteins by centrifugation. An aliquot of the soluble fraction of the cells was loaded on a NUP AGE™ Bis Tris 4-12% gradient SDS-Page (in MES) Gel (InVitrogen, Carlsbad, CA), and the gel was run according to the manufacturer's instructions.

[0146] Proteins were transferred from the gel to a nitrocellulose filter according to the NUP AGE™ SDS-polyacrylamide gel instructions. The WESTERNPRO™ AP kit (Gene Therapy Systems, San Diego, CA) was used for subsequent immunoblotting and detection according to the manufacturer's instructions. The primary antibody used for the western blots was anti 6x His (SIGMA; St. Louis, MO). The results are shown in Figure 1. No detectable VVI-A9(His₆) was seen in the soluble fraction of host cells grown in the absence of a sublethal amount of antibiotics ("Untreated", Fig.3). Soluble protein was produced by host cells that were contacted spectinomycin at different concentrations.

Example 3: Sublethal Amounts of Chloramphenicol Increases the Solubility of a HJs₆ Tagged Vaccinia Virus 1 A9 Fusion Protein in E. coli

[0147] The following Example describes a method of increasing the level of soluble recombinant protein in E. coli using a sublethal amount of chloramphenicol.

[0148] The recombinant E. coli BL21(DE3) cells described in Example 2 were cultured in 2 ml LB supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 30⁰C, shaking. The following morning, the overnight culture was diluted 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, and 200 μg/ml IPTG, to induce expression of the recombinant protein. Parallel cultures were inoculated by diluting the overnight culture 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, 200 μg/ml IPTG, and 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, or 10.0 μg/ml chloramphenicol. [0149] The cultures were grown for 12-16 hours, at 3O⁰C. The cells from 1 ml of each culture were harvested by centrifugation. The cells were resuspended in lOOμl Tris buffer, and sonicated using a Branson sonifier. Insoluble proteins were isolated from soluble proteins by centrifugation. An aliquot of the soluble fraction of the cells was loaded on a NUP AGE™ 4-12% gradient SDS-Page Gel (InVitrogen, Carlsbad, CA), and the gel was run according to the manufacturer's instructions.

[0150] Proteins were transferred from the gel to a nitrocellulose filter according to the NUP AGE™ SDS-polyacrylamide gel instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA) was used for subsequent immunoblotting and detection according to the manufacturer's instructions. The primary antibody used for the western blots was anti 6x His (SIGMA; St. Louis, MO). The results are shown in Figure 2. No detectable VVI-A9(His₆) was seen in the soluble fraction of host cells grown in the absence of a sublethal amount of antibiotics ("Untreated", Fig.3). A significant amount of Wl- A9(His₆) was detected in the soluble fraction of host cells grown in LB medium supplemented with 0.2, 0.5, 1.0, or 2.0 μg/ml chloramphenicol.

Example 4: Sublethal Amounts of Erythromycin Increases the Solubility of a Hisg Tagged

Vaccinia Virus 1 A9 Fusion Protein in E. coli

[0151] The following Example describes a method of increasing the level of soluble recombinant protein in E. coli using a sublethal amount of erythromycin.

[0152] The recombinant E. coli BL21(DE3) cells described in Example 2 were cultured in 2 ml LB supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 30⁰C, shaking. The following morning, the overnight culture was diluted 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, and 200 μg/ml IPTG, to induce expression of the recombinant protein. Parallel cultures were inoculated by diluting the overnight culture 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, 200 μg/ml IPTG, and 1.0, 2.0, 5.0, 10.0, 20.0, 35.0, or 50.0 μg/ml erythromycin.

[0153] The cultures were grown for 12-16 hours, at 30⁰C. The cells from 1 ml of each culture were harvested by centrifugation. The cells were resuspended in lOOμl Tris buffer, and sonicated using a Branson sonifier. Insoluble proteins were isolated from soluble proteins by centrifugation. An aliquot of the soluble fraction of the cells was loaded on a NUP AGE™ 4-12% gradient SDS-Page Gel (InVitrogen, Carlsbad, CA), and the gel was run according to the manufacturer's instructions.

[0154] Proteins were transferred from the gel to a nitrocellulose filter according to the NUPAGE™ SDS-polyacrylamide gel instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA) was used for subsequent irnmunoblotting and detection according to the manufacturer's instructions. The primary antibody used for the western blots was anti 6x His (SIGMA; St. Louis, MO). The results are shown in Figure 3. No detectable VVI-A9(His₆) was seen in the soluble fraction of host cells grown in the absence of a sublethal amount of antibiotics (untreated). A significant amount of VVl- A9(His₆) was detected in the soluble fraction of host cells grown in LB medium supplemented with 5.0, 10.0, 20.0, 35.0, and 50.0 μg/ml erythromycin Example 5: Sublethal Amounts of Erythromycin Increases the Solubility of a Hisg Tagged

Vaccinia Virus 1A8 Fusion Protein in E. coli

[0155] The following Example describes a method of increasing the level of soluble recombinant protein in E. coli using a sublethal amount of erythromycin.

[0156] The recombinant E. coli BL21(DΕ3) cells described in Example 2 were cultured in 2 ml LB supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 3O⁰C₅ shaking. The following morning, the overnight culture was diluted 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, and 200 μg/ml IPTG, to induce expression of the recombinant protein. Parallel cultures were inoculated by diluting the overnight culture 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, 200 μg/ml IPTG. 100 or 125 μg/ml erythromycin was added to the cultures, as indicated, when the cultures reached the indicated density, as measured by OD₆₀₀.

[0157] The cultures were grown for 12-16 hours, at 3O⁰C. The cells from 1 ml of each culture were harvested by centrifugation. The cells were resuspended in lOOμl Tris buffer, and sonicated using a Branson sonifier. Insoluble proteins were isolated from soluble proteins by centrifugation. An aliquot of the soluble fraction of the cells was loaded on a NUPAGE™ 4-12% gradient SDS-Page Gel (InVitrogen, Carlsbad, CA), and an aliquot of the insoluble fraction was loaded on a parallel gel. The gels were run according to the manufacturer's instructions.

[0158] Proteins were transferred from the gels to nitrocellulose filters according to the NUPAGE™ SDS-polyacrylamide gel instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA) was used for subsequent irnrmmoblotting and detection according to the manufacturer's instructions. The primary antibody used for the western blots was anti 6x His (SIGMA; St. Louis, MO). The results are shown in Figure 6. No detectable VVI-A9(His₆) was seen in the soluble fraction of host cells grown in the absence of a sublethal amount of antibiotics (untreated). A significant amount of Wl- A8 (His₆) was detected in the soluble fraction of host cells grown in LB medium supplemented with 5.0, 10.0, 20.0, 35.0, and 50.0 μg/ml erythromycin

Example 6: Treatment of E. coli with Elevated NaCl and Sublethal Amounts of Erythromycin Increases the Solubility of a Hisή Tagged Vaccinia Virus A9 Fusion Protein

[0159] The following Example describes a method of increasing the level of soluble recombinant protein in E. coli using a sublethal amount of Erythromycin, and culturing the cells in high salt.

[0160] The recombinant E. coli BL21(DE3) cells described in Example 2 were cultured in 2 ml LB supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 30⁰C, shaking. The following morning, the overnight culture was diluted 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, containing either no salt or 30OmM, 40OmM or 60OmM NaCl. 200 μg/ml IPTG was added to induce expression of the recombinant protein. Parallel cultures were inoculated by diluting the overnight culture 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, 200 μg/ml IPTG, and 10.0, 20.0, 35.0 or 50.0 μg/ml erythromycin no salt or 30OmM, 40OmM or 60OmMNaCl.

[0161] The cultures were grown for 12-16 hours, at 30⁰C. The cells from 1 ml of each culture were harvested by centrifugation. The cells were resuspended in lOOμl Tris buffer, and sonicated using a Branson sonifier. Insoluble proteins were isolated from soluble proteins by centrifugation. An aliquot of the soluble fraction of the cells was loaded on a NUPAGE™ 4-12% gradient SDS-Page Gel (InVitrogen, Carlsbad, CA), and the gel was run according to the manufacturer's instructions. [0162] Proteins were transferred from the gel to a nitrocellulose filter according to the NUPAGE™ SDS-polyacrylamide gel instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA) was used for subsequent immunoblotting and detection according to the manufacturer's instructions. The primary antibody used for the western blots was anti 6x His (SIGMA; St. Louis, MO). The results are shown in Figure 7. No detectable VVI-lA8(His₆) was seen in the soluble fraction of host cells grown in the absence of a sublethal amount of antibiotics (untreated). A significant amount of VVl- lA8(His₆) was detected in the soluble fraction of host cells grown in LB medium supplemented with erythromycia In some cases, the cultures grown in 30OmM NaCl produced more soluble recombinant protein than the cultures grown without elevated levels of salt.

Example 7: Sublethal Amounts of Kanamycin Increases the Solubility of a His^ Tagged Vaccinia Virus A9 Fusion Protein in E. coli

[0163] The following Example describes a method of increasing the level of soluble recombinant protein in E. coli using a sublethal amount of kanamycin.

[0164] The recombinant E. coli BL21(DE3) cells described in Example 2 were cultured in 2 ml LB supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 30⁰C, shaking. The following morning, the overnight culture was diluted 1/100 into 2 ml LB medium supplemented with 100 μg/ml carbenecillin, and 200 μg/ml IPTG, to induce expression of the recombinant protein. Parallel cultures were inoculated by diluting the overnight culture 1/100 into 2 ml LB medium supplemented with 100 μg/ml caibenecillin, 200 μg/ml IPTG, and .01, 0.2, 0.5, 1.0, 2.0, 5,0, 10.0, or 25.0 μg/ml kanamycin.

[0165] The cultures were grown for 12-16 hours, at 3O⁰C. The cells from 1 ml of each culture were harvested by centrifugation. The cells were resuspended in lOOμl Tris buffer, and sonicated using a Branson sonifier. Insoluble proteins were isolated from soluble proteins by centrifugation. An aliquot of the soluble fraction of the cells was loaded on a NUPAGE™ 4-12% gradient SDS-Page Gel (InVitrogen, Carlsbad, CA), and the gel was run according to the manufacturer's instructions. [0166] Proteins were transferred from the gel to a nitrocellulose filter according to the NUP AGE™ SDS-polyacrylamide gel instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA) was used for subsequent imrnunoblotting and detection according to the manufacturer's instructions. The primary antibody used for the western blots was anti 6x His (SIGMA; St. Louis, MO). The results are shown in Figure 4. No detectable VVI-lA9(His₆) was seen in the soluble fraction of host cells grown in the absence of a sublethal amount of antibiotics ("Untreated", Fig.3). A significant amount of VVl- lA9(His₆) was detected in the soluble fraction of host cells grown in LB medium supplemented with 0.2, 0.5, 1.0, 2.0, and 5.0 μg/ml kanamycin.

Example 8: Stepwise Addition of Sublethal Amounts of Spectmomycin Increases the Solubility of a Hisg Tagged Ketosteroid Isomerase Fusion Protein in E. coli

[0167] The following example describes a method of increasing the level of soluble recombinant protein in E. coli using a stepwise addition of sublethal amounts of spectmomycin. A pET31b plasmid (Novagen, Madison, WI) containing a C-terminal HiS₆ fusion to the ketosteroid isomerase gene (KSI) was transformed into competent E. colt BL21 (DE3) (Novagen, Madison, WI), according to the manufacturer's instructions. A control strain was constructed by transforming pET31b alone into BL21(DE3). Recombinant host cells were selected by plating the transformation on LB agar plates (See, Ausubel et ah, supra), supplemented with 100 μg/ml carbenecillin.

[0168] A carbenecillin resistant colony from each transformation reaction was isolated and used to inoculate a 2 ml culture of LB medium supplemented with 100 μg/ml carbenecillin. The cultures were grown overnight at 30⁰C, shaking. The following morning, the overnight cultures were diluted 1/50 into 35 ml LB medium supplemented with 100 μg/ml carbenecillin, and 200 μg/ml IPTG. Parallel cultures with strains harboring the plasmids for recombinant protein expression were inoculated by diluting the overnight culture 1/50 into 35 ml LB medium supplemented with 100 μg/ml carbenecillin, 200 μg/ml IPTG, and lOμg/ml, 20 μg/ml, 35 μg/ml, or 50 μg/ml spectinomycin. Another culture was started by diluting the overnight culture of the strain harboring the ρET31b ketosteroid isomerase expression plasmid 1/50 into 35 ml LB medium supplemented with 100 μg/ml carbenecillin and 200 μg/ml IPTG, and 5 μg/ml spectinomycin. Another 5 μg/ml spectinomycin was added to the culture every 51 minutes, for a total of 7 additions. The culture is referred to as Ramp 1. Another culture was started by diluting the overnight culture of the strain harboring the ρET31b ketosteroid isomerase expression plasmid 1/50 into 35 ml LB medium supplemented with 100 μg/ml carbenecillin and 200 μg/ml IPTG₅ and 10 μg/ml spectinomycin. Another 10 μg/ml spectinomycin was added to the culture every 48 minutes thereafter for a total of 5 additions. The culture is referred to as Ramp 2. A final culture was grown under conditions identical to Ramp 2, except the media was also supplemented with 35OmM NaCl. The culture is referred to as Ramp 3.

[0169] The cultures were grown for 16-18 hours, at 30⁰C. The cells from each culture were harvested by centrifugation and the cell pellets were frozen at -80⁰C. Frozen cell pellets were resuspended in 2 mis BUGBUSTER™ Protein Extraction Reagent (Novagen, Madison, WI), and incubated with agitation for 20 minutes at room temperature on a Labline Maxi Rotator. The cell lysates were clarified by centrifugation at 4oC for 30 minutes at 14,000 rpm. NiNTA Magnetic Beads (Promega, Madison, WI) were used to isolate the recombinant protein from the clarified lysates under non-denaturing conditions, using a MPC™-S magnet (Dynal), according to the manufacturer's instructions.

[0170] To measure the amount of recovered protein, 10 μl of the recovered protein was analyzed using the MICRO BCA™ protein assay kit according to the manufacturer's instructions. The results of the protein assay are depicted in Figure 5.

[0171] An aliquot of the soluble fraction of the cells was loaded on a NUP AGE™ Bis Tris 4-12% gradient SDS-Page (in MES) Gel (InVitrogen, Carlsbad, CA), and the gel was run according to the manufacturer's instructions. The gel was stained using the Colloidal Blue Stain Kit (InVitrogen, Carlsbad, CA) to visualize the proteins. The results are presented in Figure 3. Cells grown under Ramp 3 conditions produced a significant amount of soluble recombinant ketosteroid isomerase fusion protein. Figure 3, Ramp 3. Significant levels of recombinant ketosteroid isomerase were not recovered from the soluble fraction of cells that were grown in the presence of 10, 20, 35, or 50 μg/ml spectinomycin, or that were grown under Ramp 1 or Ramp 2 conditions. Example 9: Stepwise Addition of Sublethal Amounts of Spectinomycin and/or Erythromycin Increases the Solubility and Activity of a Hiss Tagged Ketosteroid Isomerase Luciferase

Fusion Protein in E. coli

[0172] The following example describes a method of increasing the level of activity of an otherwise insoluble recombinant protein in E. coli using a stepwise addition of spectinomycin, erythromycin, or a stepwise addition of a combination of spectinomycin and erythromycin. A pET31b plasmid (Novagen, Madison, WT) containing a C-terminal His₆ fusion to ketosteroid isomerase gene (KSI)/luciferase was transformed into competent E. coli BL21 (DE3) (Novagen, Madison, WI), according to the manufacturer's instructions.

[0173] A carbenecillin resistant colony from the transformation reaction was isolated and used to inoculate a 2 ml culture of LB medium supplemented with 100 μg/ml carbenecillin. The cultures were grown overnight at 30⁰C, shaking. The following morning, the overnight cultures were diluted either 1/25 or 1/50 into 5 ml LB medium to generate parallel cultures supplemented with 100 μg/ml carbenecillin. IPTG was added to a final concentration of 20OmM to some of the cultures at the time the overnight cultures were diluted, as indicated as "no recovery," in Figures 14 and 15. Other cultures were supplemented with 20OmM IPTG either 30 minutes or 60 minutes following dilution of the overnight culture, as indicated by "30 minute recovery" and "60 minute recovery," respectively, in Figures 14 and 15.

[0174] Doses of erythromycin, spectinomycin, or a combination of a spectinomycin and erythromycin were administered to the host cells approximately every 60 minutes. The amount that each dose increased the final concentration of the antibiotic is indicated in Figures 14 and 15 as follows: S 5 corresponds to an increase in the final concentration of spectinomycin by 5 μg/ml; S7.5 and SlO correspond to an increase in the final concentration of spectinomycin by 7.5 μg/ml and 10 μg/ml, respectively. E5 corresponds to an increase in the final concentration of erythromycin by 5 μg/ml, and E7.5 and ElO correspond to an increase in the final concentration of spectinomycin by 7.5 μg/ml and lOμg/ml, respectively. M5 corresponds to an increase in final concentration of 3.0 μg/ml erythromycin and 1.0 μg/ml spectinomycin. E5/S5 corresponds to an increase in the concentration of spectinomycin and erythromycin by 5 μg/ml each.. The indicated doses of antibiotics were administered to the cells approximately every 60 minutes.

[0175] After the last dose was administered to the cell cultures, the cultures were allowed to continue to grow for 16-18 hours, at 3O⁰C. For the experiment depicted in Figure 14 labeled "Purified Protein" 2ml of the 5ml culture was harvested by centrifugation. The cell pellets were frozen at -8O⁰C. Frozen cell pellets were resuspended in 2 nils BUGBUSTER™ Protein Extraction Reagent (Novagen, Madison, WI), and incubated with agitation for 20 minutes at room temperature on a Labline Maxi Rotator. The cell lysates were clarified by centrifugation at 4oC for 30 minutes at 14,000 rpm. NiNTA Magnetic Beads (Promega, Madison, WI) were used to isolate the recombinant protein from the clarified lysates under non-denaturing conditions, using a MPC™-S magnet (Dynal), according to the manufacturer's instructions. For the experiment depicted in Figure 14 labeled "Whole Lysate," 2ml of the 5ml culture was harvested by centrifugation. The supernatant was aspirated, and lOOμl of Lysis Buffer (8M Urea; 0.01M Tris-HCl, pH 8.0 and 1 tablet Complete EDTA-free protease inhibitor (Roche) per 10 ml lysis buffer) was added to the cell pellets. The tubes were incubated on ice for 10 minutes, vortexed for 30 seconds and incubated on ice for 10 minutes.

[0176] lOμl samples from the purified protein and whole lysate samples above were loaded on 4-12% gradient NuPage SDS PAGE gels (InVitrogen, Carlsbad, CA), according to the manufacturer's instructions. Proteins were transferred from the gel to a nitrocellulose filter according to the NuPage manufacturer's instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA)₅ was used for subsequent immunoblotting and detection according to the manufacturer's instructions. The primary antibody was anti 6xHis monoclonal (SIGMA; St. Louis, MO). The results are presented in Figure 14. No detectable recombinant protein was recovered from cultures that had not been treated with erythromycin and/or spectinomycin (lanes 5-6, "Purified Protein."). Recombinant protein was recovered from the soluble fraction of the cell lysates of cultures treated with sublethal concentrations of spectinomycin and/or erythromycin (lanes 1-4, "Purified Protein"). The level of recombinant protein present in whole cell lysates from cell cultures treated with sublethal concentrations of spectinomycin and/or erythromycin was lower than the level of recombinant protein present in whole cell lysates from cell cultures that had not been treated with spectinomycin or erythromycin (lanes 1-6, "Whole Lysate").

[0177] For the Experiments in Figure 15, labeled "Soluble Fraction" 2ml of the 5ml culture was harvested by centrifugation. The cells were resuspended in lQOμl Tris buffer, and sonicated using a Branson sonifier. Insoluble proteins were isolated from soluble proteins by centrifugation. For the Experiments in Figure 15 labeled "Whole Lysate," 2ml of the 5ml culture was harvested by cehtrifugation. The supernatant was aspirated, and lOOμl of Lysis Buffer (8M Urea; 0.01M Tris-HCl, pH 8.0 and 1 tablet Complete EDTA-free protease inhibitor (Roche) per 10 ml lysis buffer) was added to the cell pellets. The tubes were incubated on ice for 10 minutes, vortexed for 30 seconds and incubated on ice for 10 minutes. lOμl of each of the samples prepared for the "Soluble Fraction" and "Whole Lysate" were loaded on 4-12% gradient NuPage SDS PAGE gels (InVitrogen, Carlsbad, CA), according to the manufacturer's instructions. The Colloidal Blue Staining kit (InVitrogen, Carlsbad, CA), was used to visualize all proteins in the samples. The gel was treated according to the manufacturer's instructions. The arrow indicates the recombinant protein in the experiments labeled "Soluble Fraction" and "Whole Lysate." No recombinant protein was detected in the soluble fraction of cell lysates of cells cultured without spectinomycin and/or erythromycin ("Soluble Fraction," Lanes 6-7). Recombinant protein was detected in the soluble fraction of cell lysates of cells cultured with sublethal amounts of spectinomycin and/or erythromycin ("Soluble Fraction," Lanes 1-5). The amount of recombinant protein in the soluble fraction compared to the amount of recombinant protein in whole lysates was greater in cells cultured with spectinomycin and/or erythromycin compared to cells that were cultured without spectinomycin and/or erythromycin.

[0178] For the Experiments in Figures 14 and 15 labeled "Luciferase Assay," a Luciferase Assay kit (Stratagene, La Jolia, CA) was used to assay the specific activity of the recombinant luciferase protein from the cultures grown above. lOμl of the cell cultures were harvested and resuspended in 30μl Luciferase cell culture lysis buffer (Stratagene, La Jolla, CA). The luciferase assay was performed according to the manufacturer's instructions in a 96 well plate, and read in a luminometer. The specific activity of the luciferase fusion protein was low in whole cell lysates of cells cultured without spectinomycin and/or erythromycin (Figure 14, Columns 5-6, "Luciferase Activity"), despite the fact that an abundance of recombinant protein was present as indicated by the western blot (Figure 14, Lanes 5-6, "Whole Lysate"). On the other hand, the specific activity of the luciferase fusion protein was significantly higher in the whole cell lysates of cell cultures that had been treated with erythromycin and/or spectinomycin than those that had not been treated. (Figure 14, Lanes 1- 4, "Luciferase Assay"), even though the levels of recombinant protein present in the whole cell lysates was lower than the cultures that were untreated. (Figure 14, Lanes 1-4 "Whole Lysate"). The specific activity of the luciferase recombinant protein was increased in cell cultures treated with erythromycin and/or spectinomycin (Figure 15, "Luciferase Activity," Lanes 1-5) compared to cell cultures that were not treated with erythromycin and/or spectinomycin, (Figure 15, "Luciferase Activity," Lanes 6-8).

Example 10: General Protocol for Enrichment of Recombinant Proteins Expressed in E. coli [0179] Well known recombinant DNA techniques, as described in Ausubel et al., supra, are used to clone the gene encoding the protein of interest into an expression vector that facilitates inducible expression of the recombinant protein. The recombinant expression vector is used to transform an E. coli strain.

[0180] Following transformation, the cells are plated on selective media to isolate recombinant host cells. An isolated recombinant colony is used to inoculate 2ml LB media (see Ausubel et al., supra), supplemented with the same antibiotic used to select for recombinant host cells. The culture is grown for approximately 12-16 hours at 3O⁰C, shaking. The culture is diluted 1/200 into LB supplemented with the same antibiotics as the 2ml culture, and with an amount of inducer sufficient to induce expression of the recombinant protein. The culture is grown at 3O⁰C, shaking.

[0181] When the -OD₆₀₀ of the culture reaches approximately 0.16 to 0.18, spectinomycin is added to a final concentration of 50 μg/ml, and the culture is grown for approximately 12-16 hours at 3O⁰C, shaking. Cells are harvested by centrifugation, and the recombinant proteins are isolated using standard methods. The ratio of recombinant protein to endogenous host cell protein is increased . Example 11: Enrichment of Expression of TB C5(Hisg) in E. coli [0182] The gene encoding TB C5 (from Mycobacterium tuberculosis) was cloned into the Xbal and BamHI sites of pET3a (Novagen, Madison, WI), such that six histidine residues (6X His) were fused in-frame to the carboxy terminal end of TB C5. The recombinant plasmid was transformed into competent E. coli BL21(DE3) (Gene Therapy Systems, San Diego, CA) according to the manufacturer's instructions. Recombinant host cells were selected by plating the transformation on LB agar plates (see Ausubel et al., supra), supplemented with 100 μg/ml carbenecillin.

[0183] A carbenecillin resistant colony was isolated and used to inoculate a 2 ml culture in LB medium, supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 3O⁰C. The following morning, the culture was diluted 1/200 into 10OmL LB media, supplemented with 100 μg/ml carbenecillin and 200 μg/ml IPTG, to induce expression of the recombinant protein. The culture was grown at 30⁰C. At the indicated time points, 2ml aliquots were removed and transferred to culture tubes. The ODe₀₀ was measured, and 40, 50, 60, or 75 μg/ml spectinomycin was added to 2mL cultures. All cultures were grown overnight at 30⁰C. The following morning ImI of each culture was transferred to a 1.7ml microfuge tube. The cells were harvested by centrifugation. The supernatant was aspirated, and lOOμl of Lysis Buffer (8M Urea; 0.01M Tris-HCl, pH 8.0 and 1 tablet Complete EDTA- free protease inhibitor (Roche) per 10 ml lysis buffer) was added to the cell pellets. The tubes were incubated on ice for 10 minutes, vortexed for 30 seconds and incubated on ice for 10 minutes. lOμl was loaded on duplicate 4-12% gradient NuPage SDS PAGE gels (InVitrogen, Carlsbad, CA), according to the manufacturer's instructions.

[0184] One gel was used to perform a Western Blot, to visualize the level recombinant protein in -the cell lysate. Proteins were transferred from the gel to a nitrocellulose filter according to the NuPage manufacturer's instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA), was used for subsequent immunoblotting and detection according to the manufacturer's instructions. The primary antibody was anti 6xHis monoclonal (SIGMA; St. Louis, MO). [0185] The Colloidal Blue Staining Mt (InVitrogen, Carlsbad, CA), was used to visualize all proteins in the cell lysate, to assess enrichment of the recombinant protein. The gel was treated according to the manufacturer's instructions.

[0186] The results are shown in Figure 8. The lanes of the gels are labeled MW (molecular weight standard); 1-40, No spec (control with no spectinomycin added), The minutes indicate the time period that the culture was grown prior to the addition of spectinomycin. The last number in each sample lane indicates the concentration of spectinomycin added (μg/ml, final concentration). The graph indicates the Absorbance at OD₆Oo of the cultures at the timepoints indicated.

[0187] The level of recombinant protein relative to total cellular protein was increased 1 OX-100X.

Example 12: Enrichment of Expression of XWl IfHJs₆) in E. colt

[0188] The gene encoding XWl 1 (from Mycobacterium tuberculosis) was cloned into the Xbal and BamHI sites of pET3a (Novagen, Madison, WI), such that six histidine residues (6X His) were fused in-frame to the carboxy terminal end of XWI l. The recombinant plasmid was transformed into competent E. coli BL21(DE3) (Gene Therapy Systems, San Diego, CA) according to the manufacturer's instructions. Recombinant host cells were selected by plating the transformation on LB agar plates (see Ausubel et al., supra), supplemented with 100 μg/ml carbenecillin.

[0189] A carbenecillin resistant colony was isolated and used to inoculate a 2 ml culture in LB medium, supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 3O⁰C. The following morning, the culture was diluted 1/200 into 100ml LB media, supplemented with 100 μg/ml carbenecillin and 200μg/ml IPTG, to induce expression of the recombinant protein. The culture was grown at 30⁰C. At the indicated time points, 2ml aliquots were removed and transferred to culture tubes. The OD&oo was measured, and 40, 50, 60, or 75 μg/ml spectinomycin was added to 2ml cultures. All cultures were grown overnight at 3O⁰C. The following morning ImI of each culture was transferred to a 1.7ml microfuge tube. The cells were harvested by centrifugation. The supernatant was aspirated, and lOOμl of Lysis Buffer (8M Urea; 0.1M Tris-HCl, pH 7.5; and 1 tablet Complete EDTA- free protease inhibitor (Roche) per 10 ml lysis buffer) was added to the cell pellets. The tubes were incubated on ice for 10 minutes, vortexed for 30 seconds and incubated on ice for 10 minutes. lOμl was loaded on duplicate 4-12% gradient NuPage SDS PAGE gels (InVitrogen, Carlsbad, CA), according to the manufacturer's instructions.

[0190] One gel was used to perform a Western Blot, to visualize the level recombinant protein in the cell lysate, Proteins were transferred from the gel to a nitrocellulose filter according to the NuPage manufacturer's instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA), was used for subsequent immunoblotting and detection according to the manufacturer's instructions. The primary antibody was anti 6xHis (SIGMA; St. Louis, MO).

[0191] The Colloidal Blue Staining kit (InVitrogen, Carlsbad, CA), was used to visualize all proteins in the cell lysate, to assess enrichment of the recombinant protein. The gel was treated according to the manufacturer's instructions.

[0192] The results are shown in Figure 9. The lanes of the gels are labeled MW (molecular weight standard); 1-40, No spec (control with no spectinomycin added). The minutes indicate the time period that the culture was grown prior to the addition of spectinomycin. The last number in each sample lane indicates the concentration of spectinomycin added (μg/ml, final concentration). The graph indicates the Absorbance at OD_60O of the cultures at the timepoints indicated.

[0193] The level of recombinant protein relative to total cellular protein was increased 1 OX-100X.

Example 13: Enrichment of Expression of GFPfHisg) in E. coli

[0194] The gene encoding Green Fluorescent Protein was cloned into the Xbal and BamHI sites of pET3a (Novagen, Madison, WI). The recombinant plasmid was transformed into -competent E. coli BL21(DE3), E. coli BL21(DE3)ρLysE, or E. coli BL21(DE3)pLysS (Gene Therapy Systems, San Diego, CA) according to the manufacturer's instructions. Recombinant host cells were selected by plating the transformation on LB agar plates (see Ausubel et al., supra), supplemented with 100 μg/ml carbenecillin.

[0195] A carbenecillin resistant colony was isolated and used to inoculate a 2 ml culture in LB medium, supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 30⁰C. The following morning, the culture was diluted 1/200 into 100ml LB media, supplemented with 100 μg/ml carbenecillin and 200μg/ml IPTG, to induce expression of the recombinant protein. The culture was grown at 3O⁰C. At the indicated time points, 2ml aliquots were removed and transferred to culture tubes. The ODβoo was measured, and 50 or 60 μg/ml spectinomycin was added to 2mL cultures; All cultures were grown overnight at 3O⁰C. The following morning ImL of each culture was transferred to a 1.7ml microfuge tube. The cells were harvested by centrifugation. The supernatant was aspirated, and lOOμl of Lysis Buffer (8M Urea; 0.1 M Tris-HCl, pH 7.5; and 1 tablet Complete EDTA- free protease inhibitor (Roche) per 10 ml lysis buffer) was added to the cell pellets. The tubes were incubated on ice for 10 minutes, vortexed for 30 seconds and incubated on ice for 10 minutes. lOμl was loaded on duplicate 4-12% gradient NuPage SDS PAGE gels (InVitrogen, Carlsbad, CA), according to the manufacturer's instructions.

[0196] One gel was used to perform a Western Blot, to visualize the level recombinant protein in the cell lysate. Proteins were transferred from the gel to a nitrocellulose filter according to the NuPage manufacturer's instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA), was used for subsequent immunoblotting and detection according to the manufacturer's instructions. The primary antibody was anti-GFP (Beckton Dickinson, San Jose, CA).

[0197] The Colloidal Blue Staining kit (InVitrogen, Carlsbad, CA), was used to visualize all proteins in the cell lysate, to assess enrichment of the recombinant protein. The gel was treated according to the manufacturer's instructions.

[0198] The results are shown in Figure 10. The sample lanes are labeled with the time period (minutes) that the culture was grown prior to the addition of spectinomycin. The absorbance reading at OD₆oo is indicated. The last number in each sample lane indicates the concentration of spectinomycin added (μg/ml, final concentration).

[0199] The level of recombinant protein relative to total cellular protein was increased 1 OX-100X.

Example 14: Enrichment of Expression of VVl A9(Hisή) in E. coli

[0200] The gene encoding VVl A9 (Vaccinia Virus) was cloned into the Xbal and BamHI sites of pET3a (Novagen, Madison, WI) such that six histidine residues (6X His) were fused in-frame to the carboxy terminal end of Wl. The recombinant plasmid was transformed into competent E. coli BL21(DE3) (Gene Therapy Systems, San Diego, CA) according to the manufacturer's instructions. Recombinant host cells were selected by plating the transformation on LB agar plates (see Ausubel et al., supra), supplemented with 100 μg/ml carbenecillin.

[0201] A carbenecillin resistant colony was isolated and used to inoculate a 2 ml culture in LB medium, supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 3O⁰C. The following morning, the culture was diluted 1/200 into 10OmL LB media, supplemented with 100 μg/ml carbenecillin and 200μg/ml IPTG, to induce expression of the recombinant protein. The culture was grown at 3O⁰C. At the indicated time points, 2ml aliquots were removed and transferred to culture tubes. The OD_δoo was measured, and 50 or 60 μg/ml spectinomycin was added to 2mL cultures. All cultures were grown overnight at 30⁰C. The following morning ImI of each culture was transferred to a 1.7mL microfuge tube. The cells were harvested by centrifugation. The supernatant was aspirated, and lOOμL of Lysis Buffer (8M Urea; 0.1M Tris-HCl, pH 7.5; and 1 tablet Complete EDTA- free protease inhibitor (Roche) per 10 ml lysis buffer) was added to the cell pellets. The tubes were incubated on ice for 10 minutes, vortexed for 30 seconds and incubated on ice for 10 minutes. lOμl was loaded on duplicate 4-12% gradient NuPage SDS PAGE gels (mVitiOgen, Carlsbad, CA), according to the manufacturer's instructions.

[0202] One gel was used to perform a Western Blot, to visualize the level recombinant protein in the cell lysate. Proteins were transferred from the gel to a nitrocellulose filter according to the NuPage manufacturer's instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA), was used for subsequent immunoblotting and detection according to the manufacturer's instructions. The primary antibody was anti 6xHis (SIGMA; St. Louis, MO).

[0203] The Colloidal Blue Staining kit (InVitrogen, Carlsbad, CA), was used to visualize all proteins in the cell lysate, to assess enrichment of the recombinant protein. The gel was treated according to the manufacturer's instructions.

[0204] The results are shown in Figure 11. The sample lanes are labeled with the time period (minutes) that the culture was grown prior to the addition of spectinomycin. The absorbance reading at ODβoo is indicated. The last number in each sample lane indicates the concentration of spectinomycin added (μg/ml, final concentration).

[0205] The level of recombinant protein relative to total cellular protein was increased 1 OX-10OX.

Example 15: Enrichment of Expression of WlA9(His₆) in E. coli With Streptomycin and

Chloramphenicol

[0206] The gene encoding VVl (Vaccinia Virus) was cloned into the Xbal and BamHI sites of pET3a (Novagen, Madison, WI) such that six histidine residues (6X His) were fused in-frame to the carboxy terminal end of VVl . The recombinant plasmid was transformed into competent E. coli BL21(DE3) (Gene Therapy Systems, San Diego, CA) according to the manufacturer's instructions. Recombinant host cells were selected by plating the transformation on LB agar plates (see Ausubel et al., supra), supplemented with 100 μg/ml carbenecillin.

[0207] A carbenecillin resistant colony was isolated and used to inoculate a 2 ml culture in LB medium, supplemented with 100 μg/ml carbenecillin. The culture was grown overnight at 3O⁰C. The following morning, the culture was diluted 1/200 into 100ml LB media, supplemented with 100 μg/ml carbenecillin and 200μg/ml IPTG, to induce expression of the recombinant protein. The culture was grown at 3O⁰C. After 3 hours, the indicated amounts of Streptomycin and Chloramphenicol were added to the 2mL cultures. All cultures were grown overnight at 3O⁰C. The following morning ImL of each culture was transferred to a 1.7ml microfuge tube. The cells were harvested by centrifugation. The supernatant was aspirated, and lOOμl of Lysis Buffer (8M Urea; 0.1M Tris-HCl, pH 7.5; and 1 tablet Complete EDTA-free protease inhibitor (Roche) per 10 ml lysis buffer) was added to the cell pellets. The tubes were incubated on ice foτ 10 minutes, vortexed for 30 seconds and incubated on ice for 10 minutes. lOμl was loaded on duplicate 4-12% gradient NuPage SDS PAGE gels (InVitrogen, Carlsbad, CA), according to the manufacturer's instructions.

[0208] One gel was used to perform a Western Blot, to visualize the level recombinant protein in the cell lysate. Proteins were transferred from the gel to a nitrocellulose filter according to the NuPage manufacturer's instructions. The WESTERNPRO™ AP kit (GeneTherapy Systems, San Diego, CA), was used for subsequent immunoblotting and detection according to the manufacturer's instructions. The primary antibody was anti 6xHis (SIGMA; St. Louis, MO).

[0209] The Colloidal Blue Staining kit (InVitrogen, Carlsbad, CA), was used to visualize all proteins in the cell lysate, to assess enrichment of the recombinant protein. The gel was treated according to the manufacturer's instructions.

[0210] The results are shown in Figures 12. The sample lanes are labeled 1-23- (first number). The next two numbers above each sample lane indicate the concentration of streptomycin and chloramphenicol, respectively, added to each culture.

[0211] The level of recombinant protein relative to total cellular protein was increased.

Example 16: Sublethal Amounts of Erythromycin or Spectinomvcin Increases the Solubility ofMMBl in £. co// DH5α

[0212] The following Example describes a method of increasing the level of soluble recombinant protein in E. coli using a sublethal amount of erythromycin or spectinomycin,

[0213] The gene encoding MMBl (mus musculus) was cloned into the pBAD expression vector (InVitrogen, Carlsbad, CA). The recombinant plasmid was transformed into competent E. coli DH5α (Gene Therapy Systems, San Diego, CA) according to the manufacturer's instructions. Recombinant host cells were selected by plating the transformation on LB agar plates (see Ausubel et al., supra), supplemented with 100 μg/ml ampicillin.

[0214] An ampicillin resistant colony was isolated and used to inoculate a 10 ml culture in LB medium, supplemented with 100 μg/ml ampicillin. The culture was grown overnight at 3O⁰C. The following morning, the culture was diluted 1/15 into 100ml LB media, supplemented with 100 μg/ml ampicillin. The culture was grown at 37⁰C until it reached an OD_60O of 0.5, at which time the culture media was supplemented with 0.1% arabinose to induce expression of the recombinant protein. As indicated in Figure 16, some cultures were supplemented with either 15, 25, 35, or 45 μg/ml erythromycin. Other cultures were treated with four different doses of either erythromycin or spectinomycin, increasing the final concentration of antibiotics in the culture media by 10 μg/ml/dose. For cultures treated with four doses of antibiotics, the first dose was administered at the time of induction and either at 25 minute intervals or 40 minute intervals, as indicated. The cultures were all grown for 5 hours at 37⁰C following induction with arabinose.

[0215] 2mL of each culture was transferred to a 1.7ml microfuge tube. The cells were harvested by centrifugation. The cells were resuspended in lOOμl Tris buffer, and sonicated using a Branson sonifier. Insoluble proteins were isolated from soluble proteins by centrifugation. An aliquot of the soluble fraction of the cells was loaded on a NUP AGE™ 4- 12% gradient SDS-Page Gel (InVitrogen, Carlsbad, CA), and the gel was run according to the manufacturer's instructions.

[0216] The Colloidal Blue Staining kit (InVitrogen, Carlsbad, CA), was used to visualize all proteins in the cell lysate, to assess expression of the recombinant protein in the insoluble fraction or the total cell lysate. The gel was treated according to the manufacturer's instructions.

[0217] The results are shown in Figure 16. The sample lanes are labeled 1-11. Lanes 1-4 are the soluble fractions from cells treated with multiple additions of spectinomycin (1 and 3) spaced at 25 (lane 1) or 40 minutes (lane 2) or erythromycin (2 and 4) spaced 25 (lane 2) or 40 minutes (lane 4) apart. Lanes 5-8 are single additions of erythromycin; 15ug/ml, 25 μg/ml, 35 μg/ml and 40 μg/ml respectively. Lane 9 is the soluble fraction from the untreated culture induced with arabinose. Lane 10 is the total protein lysate from the same cells as lane 9. Lane 11 is the total protein from the uninduced control cells. Example 17: Increased Solubility and Enrichment of Recombinant Protein Expression in B. subtilis

[0218] Well known recombinant DNA techniques, as described in Ausubel et al., supra, are used to clone the gene encoding the protein of interest into an expression vector that facilitates inducible expression of the recombinant protein. The recombinant expression vector is used to transform a B. subtilis strain.

[0219] Following transformation, the cells are plated on selective media to isolate recombinant host cells. An isolated recombinant colony is used to inoculate 2ml LB media (see Ausubel et al., supra), supplemented with the same antibiotic used to select for recombinant host cells. The culture is grown for approximately 12-16 hours at 3O⁰C, shaking. The culture is diluted 1/200 into LB supplemented with the same antibiotics as the 2ml culture, and with an amount of inducer sufficient to induce expression of the recombinant protein. The culture is grown at 3O⁰C, shaking.

[0220] When the OD_60O of the culture reaches approximately 0.16 to 0.18, spectinomycin is added to a final concentration of 50 μg/ml, and the culture is grown for approximately 12-16 hours at 3O⁰C, shaking. Cells are harvested by centrifugation, and the recombinant proteins are isolated using standard methods. The level of recombinant protein in the soluble fraction of the cells is increased. The ratio of recombinant protein to endogenous host cell protein is increased.

Example 18: Increased Solubility and Enrichment of Recombinant Protein Expression in Yeast cells with a Heterologous RNA Polymerase

[0221] The gene encoding the protein of interest is cloned into a yeast expression vector such as pYES vector (InVitrogen, Carlsbad, CA) that contains a tryptophan biosynthetic gene to ensure selective propagation in a host cell lacking this gene product. The T7 RNA polymerase gene (preceded by the Gal promoter) is cloned into a different pYES vector that contains a uracil biosynthetic gene on the plasmid. Saccharomyces cerivisiae is transformed with lμg of each recombinant plasmid. A colony capable of growth on media lacking both tryptophan and uracil is isolated and used to inoculate a 10 ml culture in minimal media. The culture is grown for approximately 16 hours at 25⁰C. The culture is diluted 1/200 in identical media with galactose as a carbon source and grown for approximately 6 hours at 25⁰C. A sublethal concentration of cycloheximide is added to the culture. The culture is grown for approximately 16 hours at 25⁰C. The cells are harvested by centrifugation and the recombinant protein is isolated using standard techniques. The ratio of recombinant protein to endogenous host cell protein is increased. Example 19: Increased Solubility and Enrichment of Recombinant Protein Expression in

Yeast cells

[0222] The gene encoding the protein of interest is cloned into a yeast expression vector. A yeast host strain is transformed with Iμg of each recombinant plasmid using standard methods as described in Ausubel et al., supra. A recombinant colony is isolated and used to inoculate a 10 ml culture in minimal media. The culture is grown for approximately 16 hours at 25⁰C. The culture is diluted 1/200 in identical media supplemented with an inducer to induce expression of the recombinant protein. The culture is grown for approximately 6 hours at 25⁰C. A sublethal concentration of a compound that inhibits macromolecular synthesis is added to the culture. The culture is grown for approximately 16 hours at 25⁰C. The cells are harvested by centrifugation and the recombinant protein is isolated using standard techniques. The level of recombinant protein in the soluble fraction of the cells is increased. The ratio of recombinant protein to endogenous host cell protein is increased.

Example 20: Increased Solubility and Enrichment of Recombinant Protein Expression in

Mammalian cells

[0223] The gene encoding the protein of interest is cloned into a mammalian expression vector such as the phCMV vector, that includes the promoter for T7 upstream of the coding region and the T7 RNA polymerase gene driven by the CMV promoter. The construct is used to transfect a mammalian cell line such as NIH3T3 using standard protocols.

[0224] Alternatively, the gene encoding the protein of interest is cloned into the phCMV vector (under the control of the T7 promoter). The gene encoding T7 RNA polymerase is cloned into a viral vector, such as a Vaccinia Virus expression vector as described in Huemer, H.P. et. Al. J. Virol. Methods 85:1 (2000), which is herein incorporated by reference in its entirety. A mammalian cell line is co-transfected with the plasmid and virus, using standard protocols.

[0225] Alternatively, the gene encoding the protein of interest is cloned into a mammalian expression vector such as the phCMV vector such that the gene is transcribed from a T7 promoter. This construct is used to transfect a stable mammalian cell line that carries the T7 RNA polymerase gene.

[0226] Following transfection, cells are grown for 12-24 hours at 37⁰C. A sublethal amount of an inhibitor of macromolecular synthesis is added to the cells. Cells are grown for an additional 24-48 hours at 37⁰C. Cells are harvested and the recombinant protein is isolated according to standard protocols. See Ausubel et al., supra. The level of recombinant protein in the soluble fraction of the cells is increased. The ratio of recombinant protein to endogenous host cell protein is increased. Example 21 : Increased Solubility and Enrichment of Recombinant Protein Expression in

Insect cells

[0227] The gene encoding the recombinant protein of interest is cloned into a Baculovirus expression vector, such as the pBAC vector (InVitrogen, Carlsbad, CA).

[0228] Insect host cells are co-transfected with the plasmids according to standard procedures set forth in Ausubel et al., supra, or according to manufacturer's instructions. Cells are grown under conditions to allow expression of the recombinant protein. A sublethal concentration of a compound that reduces host cell macromolecular synthesis is added to the culture. Cells were grown for 48-162 hours at 37⁰C. The cells are harvested and the proteins are isolated according to standard protocols. See, e.g., Ausubel et al., supra, which is herein incorporated by reference in its entirety. The level of recombinant protein in the soluble fraction of the cells is increased. The ratio of recombinant protein to endogenous host cell protein is increased. Example 22: A General Method for Determining a Sublethal Concentration of Antibiotics

Suitable for Recombinant Protein Expression in Bacteria

[0229] For bacteria other than E. coli, the methods described in Examples 1-5 are used to increase the solubility of recombinant proteins. To calculate the amount or concentration of antibiotics that is sublethal to the sensitive host cell, the recombinant bacterial host cell is challenged with a range of antibiotics. The growth rate of the cells is measured by determining the optical density of the culture over time. The growth rate is compared with the growth rate of cells grown in media that is not supplemented with a sublethal amount of an antibiotic. A concentration of antibiotic is considered sublethal if there is an increase in cell density over a 24 hour period but this increase is less than the untreated cells. Example 23: A General Method for Determining a Sublethal Concentration of Antibiotics

Suitable for Recombinant Protein Expression in Yeast

[0230] To calculate the amount or concentration of antibiotics that is sublethal to the sensitive yeast host cell, the recombinant yeast cell is challenged with a range of antibiotics. The growth rate of the cells is measured by determining the optical density of the culture over time. The growth rate is compared with the growth rate of cells grown in media that is not supplemented with a sublethal amount of an antibiotic. A considered to be sublethal if there is an increase in cell density over a 24 hour period but this increase is less than the un-treated cells. Example 24: A General Method for Determining a Sublethal Concentration of Compounds

Suitable for Recombinant Protein Expression in Mammalian Cells [0231] CHOKl and 293 cells were grown in complete DMEM medium under standard conditions (See Ausubel et al, supra) in a 24 well plate. A dilution series of antibiotics were added to each well. After 24 and 48 hours, the cells were inspected visually by microscopy to determine whether they grew to confluency, were killed, or remained unchanged without concomitant cell death. Table 2 lists the range of antibiotic concentrations that did not kill the cells.

Table 2: Sublethal Concentration Ranges for Compounds Useful in Recombinant Protein

Expression in Mammalian Cells

Example 25: Expression of Recombinant Proteins in Bacteria [0232] Various E. coli strains, including BL21(DE3), BL21(DE3)pLysE and BL21(DE3)pLysS were transformed with various expression plasmids for a variety of proteins, including the following diverse proteins: VV1_A9 and VVI_A8, derived from Vaccinia Virus, TCB 5 and XW-I l, derived from Mycobacterium tuberculosis, Green Fluorescent Protein from jellyfish (Aequorea Victoria), and Ketosteriod Isomerase protein from humans. Each expression construct contained a bla gene that functioned as a selectable marker, and rendered the host cells resistant to carbenecillin at lOOμg/ml. The nucleic acids encoding the recombinant proteins were operably linked to promoters that enabled IPTG- dependent expression of the proteins. The E. coli host cells were sensitive to erythromycin, kanamycin, chloramphenicol, spectinomycin, and streptomycin.

[0233] Recombinant E coli host cells were used to inoculate a starter culture of LB media supplemented with lOOμg/ml carbenecillin. The cultures were grown for approximately 16-18 hours at 3O⁰C, shaking. The starter cultures were diluted either 1/50 or 1/100 to start parallel cultures in LB media supplemented with lOOμg/ml carbenecillin. Other parallel cultures were started in media was supplemented with 30OmM, 40OmM or 60OmM NaCl. The cultures were grown at 3O⁰C, shaking. The ODeoo of the cultures was measured. At various timepoints, recombinant protein expression was induced by adding IPTG (either 10OmM, 20OmM or 50OmM, final concentration) to the culture. At various timepoints, sublethal amounts of various antibiotics, such as erythromycin, kanamycin, chloramphenicol, spectinomycin, and streptomycin were added. In some cultures, more than one antibiotic was added, such as both chloramphenicol and spectinomycin. In some cultures, the sublethal amount of antibiotic was added stepwise, over time. The cultures were grown for another 12-18 hours at 3O⁰C, shaking.

[0234] The cells were harvested and lysed either by resuspension in a lysis buffer, or by sonication. Cells lysed with lysis buffer were loaded directly onto an SDS-PAGE gel. The Sonicated cell lysates were centrifuged to separate the soluble and insoluble fractions of the cells, and fractions of each were loaded onto an SDS-PAGE gel. Total cellular protein and recombinant proteins were visualized by Colloidal Blue staining or Western Blotting with a 6x His antibody, respectively. An increase in the ratio of recombinant protein compared to total host cell protein was observed. An increase in the amount of soluble recombinant protein was observed.

Claims

WHAT IS CLAIMED IS

1. A method of producing a recombinant protein, comprising: providing a host cell that is sensitive to a first compound, wherein the host cell comprises a recombinant nucleic acid construct, and wherein said nucleic acid construct encodes a recombinant protein; and contacting the host cell with the first compound.

2. The method of Claim 1, wherein the recombinant protein that is obtained comprises soluble recombinant protein.

3. The method of Claim 2, wherein the amount of soluble protein that is obtainable is greater than the amount of soluble protein that is obtainable from the host cell when the host cell is not contacted with the first compound.

4. The method of Claim 1, wherein the recombinant protein obtained has a greater specific activity when compared to recombinant protein not obtained according the method of Claim 1.

5. The method of Claim 1 where the amount of recombinant protein compared with the overall protein is increased.

6. The method of Claim 1, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

7. The method of Claim 6, wherein the host cell is a prokaryotic cell.

8. The method of Claim 7, wherein the prokaryotic host cell is an Escherichia coli, a Salmonella typhimurium, a member of the genus Bacillus or a member of the genus Pseudomonas.

9. The method of Claim 8, wherein the E. coli is selected from the group consisting of BL21, BL2l(DE3), DH5α, JM109, JMlOl₅ HBlOl, BL21(DE3) pLysS, BL21(DE3) pLysE, DHlOB, XL-I Blue, XL-10 Gold, NM522, and TOPlO.

10. The method of Claim 6, wherein the host cell is a eukaryotic cell.

11. The method of Claim 10, wherein the eukaryotic cell is a yeast cell.

12. The method of Claim 11, wherein the yeast cell is a Pichia pastoris cell, a Saccharomyces cerevisiae cell, or a Schizosaccharomycespom.be cell.

13. The method of Claim 10, wherein the host cell is a mammalian cell. 14, The method of Claim 13, wherein the mammalian cell is a CHO cell, a COS-7 cell, a 293 cell, or a NIH-3T3 cell. The method of Claim 10, wherein the eukaryotic cell is an insect cell.

16. The method of Claim 15, wherein the insect cell is an Sf-9 cell

17. The method of Claim 1, wherein the first compound is an antibiotic or an analog of an antibiotic.

18. The method of Claim 17, wherein the antibiotic is selected from the group consisting of streptomycin, gentamycin, amikacin, kanamycin, neomycin, netilmycin, paromycin, spectinomycin, tobramycin, erythromycin, azithromycin, clarithromycin, roxithromycin, telithromycin, chloramphenicol, puromycin, cycloheximide, diphtheria toxin, G-418, hygromycin, blasticin S, and doxorubicin..

19. The method of Claim 1 , wherein the first compound inhibits translation.

20. The method of Claim 19, wherein the first compound targets the small ribosomal subunit.

21. The method of Claim 1, wherein the first compound targets the large ribosomal subunit.

22. The method of Claim 1, wherein the contacting step comprises contacting the host cell with a sub- lethal amount of the first compound.

23. The method of Claim 22, wherein the sub- lethal amount does not immediately kill the host cell.

24. The method of Claim 1, wherein the recombinant nucleic acid construct comprises a heterologous promoter operably linked to a nucleic acid encoding the recombinant protein.

25. The method of Claim 24, wherein transcription from the heterologous promoter is induced by a regulator compound.

26. The method of Claim 25 further comprising contacting the host cell with the regulator compound.

27. The method of Claim 26, wherein the host cell is contacted with the first compound after the host cell is contacted with the regulator compound. 28. The method of Claim 27, wherein the host cell is contacted with the first compound approximately 30 minutes, one hour, one and a half hours, two hours, three hours, four hours, or five hours after the host cell is contacted with the regulator compound.

29. . The method of Claim 27, wherein the host cell is contacted with the first compound at approximately the same time that the host cell is contacted with the regulator compound.

30. The method of Claim 1 , wherein more than one dose of the first compound is administered to the host cell.

31. The method of Claim 30, wherein the host cell is an E. coli cell, and wherein the first compound is spectinomycin, and wherein the concentration of spectinomycin to which the host cell is contacted increases from about 5μg/ml, to lOμg/ml, to 15μg/ml, to 20μg/ml, to 25μg/ml, to 30μg/ml, to 35μg/ml following the administration of each dose.

32. The method of Claim 24, wherein the heterologous promoter is a bacteriophage promoter.

33. The method of Claim 32, wherein transcription from the heterologous promoter is induced by a regulator compound, and wherein the regulator compound induces expression of a bacteriophage RNA polymerase that can recognize the bacteriophage promoter.

34. The method of Claim 33, wherein the bacteriophage promoter is a phage T7 promoter.

35. The method of Claim 25 , wherein the regulator compound is IPTG.

36. The method of claim 1 wherein an endogenous host cell promoter directs expression of the recombinant protein.

37. The method of Claim 36, wherein the endogenous host cell promoter is araC, and the regulator compound is arabinose.

38. The method of Claim 1, wherein the host cell further comprises a selectable marker.

39. The method of Claim 38, further comprising culturing said host cell in a media comprising a selection compound that enables only host cells comprising the selectable marker to propagate. 40. The method of Claim 1 , farther comprising maintaining the host cell to permit expression of the recombinant protein, wherein said maintaining step comprises contacting the host cell with a growth medium.

41. The method of Claim 1, further comprising contacting the host cell with an alkali metal salt or an alkaline earth metal salt.

42. The method of Claim 41 , wherein the alkali metal salt is NaCl.

43. The method of Claim 1, further comprising contacting the host cell with an osmoprotectant.

44. The method of Claim 43, wherein the osmoprotectant is selected from the group consisting of: glycine, glycine-betaine, ectoine, sugars, sugar alcohols, carnitine, proline, glutamate, sorbitol, and mannitol.

45. The method of Claim 1, further comprising contacting the cells with an oxidizing agent or an antioxidant.

46. The method of Claim 45, wherein the oxidizing agent or antioxidant is hydrogen peroxide.

47. The method of Claim 46, wherein the cells are contacted with 0.01% hydrogen peroxide.

48. The method of Claim 1 further comprising contacting the cells with a reducing agent.

49. The method_, of claim 48, wherein the cells are contacted with 2- mercaptothanol or dithiothreitol.

50. A method of producing a recombinant protein, comprising: providing an Escherichia coli (E. colϊ) host cell that is sensitive to an antibiotic selected from the group consisting of spectinomycm, erythromycin, chloramphenicol, kanamycin, or streptomycin, wherein the JS. coli host cell is a BL21 strain, and wherein the E. coli comprises a recombinant nucleic acid construct, wherein said nucleic acid construct encodes a recombinant protein; and contacting the E. coli host cell with the antibiotic.

51. A method of producing a recombinant protein in vitro, comprising: providing an in vitro transcription-translation system; providing a recombinant nucleic acid encoding a recombinant protein that is operably linked to transcriptional and translational regulatory elements recognized by the in vitro transcription-translation system; contacting the recombinant nucleic acid and the in vitro transcription translation system; and contacting the recombinant nucleic acid and the in vitro transcription translation system with an antibiotic.

52. The method of Claim 51, wherein the compound is added to the in vitro transcription-translation system before the recombinant nucleic acid is added to the in vitro transcription-translation system.

53. The method of Claim 51, wherein the compound is added to the in vitro transcription-translation system at approximately the same time as the recombinant nucleic acid is added to the in vitro transcription-translation system.

54. The method of Claim 51, wherein the first compound is an antibiotic or an analog of an antibiotic.

55. The method of Claim 54, wherein the antibiotic is selected from the group consisting of erythromycin, azithromycin, clarithromycin, roxithromycin, telithromycin, streptomycin, gentamycin, amikacin, kanamycin, neomycin, netilmycin, paromycin, spectinomycin, tobramycin, erythromycin, and chloramphenicol.

56. A method for producing a recombinant protein, comprising: providing a host cell comprising a recombinant nucleic acid construct, wherein said recombinant nucleic acid construct operably encodes said recombinant protein, and wherein said host cell further comprises a selectable marker; culturing said host cell in a media comprising a selection compound that enables only host cells comprising the . selectable marker to propagate; administering to said host cell a concentration of a first compound that reduces the production of native protein by said host cell; and thereafter harvesting said host cells and recombinant protein from said host cell.

57. A method of increasing the ratio of expressed recombinant protein to expressed host cell protein, comprising: providing a host cell, wherein the host cell comprises endogenous DNA encoding host cell protein, a recombinant nucleic acid construct encoding a recombinant protein, and a selectable marker, and wherein the host cell is sensitive to a first compound; culturing said host cell in a media comprising a selection compound that enables only host cells comprising the selectable marker to propagate, and contacting the host cell with the first compound, wherein the first compound reduces host cell protein synthesis, and wherein the ratio of expressed recombinant protein to expressed host cell protein in the host cell increases after contacting the host cell with the first compound.

58. A method of increasing the ratio of soluble recombinant protein to insoluble recombinant protein expressed in a host cell, comprising: providing a host cell, wherein the host cell comprises a selectable marker and a recombinant nucleic acid construct encoding a recombinant protein, and wherein the host cell is sensitive to a first compound; culturing said host cell in a media comprising a selection compound that enables only host cells comprising the selectable marker to propagate, and contacting the host cell with the first compound, wherein the first compound reduces host cell protein synthesis, and wherein the ratio of soluble recombinant protein to expressed to insoluble recombinant protein expressed in the host cell increases after contacting the host cell with the first compound.

59. In a method for producing a recombinant protein from a recombinant nucleic acid construct in a host cell, wherein the host cell is grown in media comprising a selection compound, and wherein the host cell comprises a selectable marker, the improvement comprising reducing the level of background protein expression by administering to the host cell prior to harvesting the recombinant protein a sublethal concentration of a first compound to which the host cell is sensitive, wherein the first compound is a reducer of native protein synthesis by said host cell.

60. In a method for producing a recombinant protein from a recombinant nucleic acid construct in a host cell, wherein the host cell is grown in media comprising a selection compound, and wherein the host cell comprises a selectable marker, the improvement comprising increasing the level of soluble recombinant protein expressed, by administering to the host cell prior to harvesting the recombinant protein a sublethal concentration of a first compound to which the host cell is sensitive, wherein the first compound is a reducer of native protein synthesis by said host cell. 61. A method of increasing the level of a recombinant protein relative to the level of cellular protein in a host cell comprising:

(i) contacting a host cell with a regulator compound, wherein said regulator compound induces the expression of a recombinant protein, and

(ii) contacting the host cell with a composition comprising a sublethal concentration of a first compound, wherein said first compound causes a decrease in the expression of cellular protein as compared to a host cell that has not been contacted with said first compound, wherein the host cell is sensitive to the first compound.

62. A method of increasing the level soluble recombinant protein relative to the level insoluble recombinant protein expressed in a host cell comprising:

(ii) contacting the host cell with a composition comprising a sublethal concentration of a first compound, wherein said first compound causes an increase in the level of soluble recombinant protein as compared to a host cell that has not been contacted with said first compound, wherein the host cell is sensitive to the first compound.

63. A method of increasing the ratio of expressed recombinant protein to expressed host cell protein, comprising: providing a host cell, the host cell comprising endogenous DNA encoding host cell protein and a recombinant nucleic acid construct encoding a recombinant protein, wherein the host cell is sensitive to a first compound; and contacting the host cell with the first compound, wherein the first compound reduces host cell macromolecular synthesis, and wherein the ratio of expressed recombinant protein to expressed host cell protein in the host cell increases after contacting the host cell with the first compound.

64. A method for producing a recombinant protein from a recombinant nucleic acid construct in a host cell, the improvement comprising reducing the level of background protein expression by administering to the host cell a sublethal concentration of a first compound, wherein the first compound reduces host macromolecular synthesis prior to harvesting the recombinant protein.

65. A method for producing a recombinant protein, comprising: providing a host cell having a recombinant nucleic acid construct, wherein said recombinant nucleic acid construct operably encodes said recombinant protein; administering to said host cell a sublethal concentration of a first compound that reduces synthesis of native macromolecules by said host cell.

66. A method of increasing the ratio of expressed recombinant protein to expressed host cell protein, comprising: providing a host cell, the host cell comprising endogenous DNA encoding host cell protein and a recombinant nucleic acid construct encoding a recombinant protein, wherein said host cell is sensitive to a first compound, and wherein the recombinant nucleic acid construct further renders the host cell insensitive to a second compound, contacting the host cell with the first compound, wherein the first compound reduces host cell macromolecular synthesis, and wherein the ratio of expressed recombinant protein to expressed host cell protein in the host cell increases after contacting the host cell with the first compound, and contacting the host cell with the second compound.

67. A method for producing a recombinant protein, comprising: providing a population of cells, wherein said population comprises one or more cells that are susceptible to a first compound and one or more host cells having a recombinant nucleic acid construct, wherein said recombinant nucleic acid construct confers resistance against the first compound, and wherein said recombinant nucleic acid construct operably encodes a recombinant protein; contacting said population of cells with the first compound; selecting one or more host cells having a recombinant nucleic acid construct that have been contacted -with the first

and administering a sublethal concentration of a second compound to the selected one or more host cells having a recombinant nucleic acid construct, wherein the second compound reduces the production of native protein in the host cells.

68. A method for producing a recombinant protein, comprising: providing an E. coli host cell wherein said E. coli host cell comprises an inducible T7 RNA polymerase system, and wherein the E. coli host cell further comprises a recombinant nucleic acid construct, wherein said recombinant nucleic acid construct operably encodes said recombinant protein, and wherein said recombinant nucleic acid construct comprises a T7 promoter; administering to said E, coli host cell a sublethal concentration of an antibiotic or antibiotic analog that reduces the production of native protein by said E. coli host cell; and thereafter harvesting said E. coli host cell and recombinant protein from said E. coli host cell.

69. A method of producing a soluble recombinant protein in a host cell comprising:

(i) contacting a host cell with a first compound that increases expression of the recombinant protein; and

(ii) contacting the host cell with a sublethal concentration of a second compound, wherein the sublethal concentration of the second compound causes an increase in the level of soluble recombinant protein within the host cell, wherein the host cell is sensitive to the second compound.

70. A method of producing a soluble recombinant protein within an E. coli host cell, comprising: providing an E. coli host cell, the host cell comprising a recombinant nucleic acid construct encoding a recombinant protein, wherein the host cell is sensitive to a first compound, wherein the first compound is selected from the group consisting of chloramphenicol, spectinomycin, erythromycin, and kanamycin; contacting the host cell with a first composition that induces recombinant protein expression, contacting the host cell with a second composition, wherein the second composition comprises the first compound; and a compound selected from the group consisting of an alkali salt, oxidizing agent or an antioxidant, and a reducing agent.

71. The method of Claim 70, wherein the first composition and the second composition are administered together.

72. The method of Claim 71, wherein the first composition and the second composition are mixed together prior to administration.

73. The method of Claim 70, wherein the E. coft is BL21.

74. The method of Claim 70, wherein the first compound is selected from the group consisting of chloramphenicol, spectinomycin, erythromycin, and kanamycin 75. The method of Claim 74, wherein the host cell is contacted with about 0.1 to about 35 microgranis/ml of the first compound.

76. A kit for increasing the levels of a recombinant protein relative to cellular protein in a host cell comprising: a first compound that increases the ratio of recombinant protein to native host cell protein, wherein the host cell is sensitive to the first compound.

77. A kit for increasing the solubility of a recombinant protein expressed in a host cell engineered to express the recombinant protein comprising: a composition comprising a first compound, wherein the host cell is sensitive to the first compound, and wherein administering a sublethal concentration of the first compound to a host cell that is expressing the recombinant protein increases the level of soluble recombinant protein within the cell.

78. The kit of Claim 76 or 77 wherein the recombinant protein has a tag.

79. The kit of Claim 78 wherein the tag is a six-histidine tag.

80. The kit of Claim 76 or 77, further comprising an expression vector for expression of the recombinant protein.

81. The kit of Claim 80, wherein the expression vector renders the host cell resistant to a second compound.

82. The kit of Claim 76 or 77, further comprising a host cell.

83. The kit of Claim 82 wherein the host cell is competent.

84. The kit of Claim 83, wherein the competent host cell is Escherichia coli BL21(DE3).

85. The kit of Claim 76 or 77, wherein the first compound is an antibiotic selected from the group consisting of streptomycin, gentamycin, amikacin, kanamycin, neomycin, netilmycin, paromycin, spectinomycin, tobramycin, erythromycin, azithromycin, clarithromycin, roxithromycin, telithrorπycin, chloramphenicol.

86. In a method for producing a recombinant protein having limited solubility, the improvement comprising increasing the amount of soluble recombinant protein by providing a host cell that is sensitive to a first compound, wherein the host cell comprises a recombinant nucleic acid construct, and wherein said nucleic acid construct encodes a recombinant protein; and contacting the host cell with the first compound. 87. In a method for producing a recombinant protein having limited activity, the improvement comprising increasing the activity of the recombinant protein by providing a host cell that is sensitive to a first compound, wherein the host cell comprises a recombinant nucleic acid construct, and wherein said nucleic acid construct encodes a recombinant protein; and contacting the host cell with the first compound.

88. A method of increasing the specific activity of a recombinant protein obtained from a host cell, comprising: identifying a protein that has limited/lower/diminished activity when produced in an in vitro or an in vivo recombinant protein expression system; providing a host cell that is sensitive to a first compound, wherein the host cell comprises a recombinant nucleic acid construct, and wherein said nucleic acid construct encodes the protein; and contacting the host cell with the first compound.

89. The method of Claims 87 or 88, wherein the recombinant protein obtained has a greater specific activity when compared to recombinant protein not obtained according the method of Claim 87 or Claim 88.

90. In a method for producing a recombinant protein having limited activity, the improvement comprising increasing the activity of the recombinant protein by providing an expression system that is sensitive to a first compound, wherein the expression system comprises a recombinant nucleic acid construct, and wherein said nucleic acid construct encodes a recombinant protein; and contacting the expression system with the first compound.

91. The method- of Claim 90, wherein the expression system is an in vitro expression system.

92. The method of Claim 90, wherein the expression system is an in vivo expression system, comprising a host cell.

93. The method of any one of Claims 1, 50, 65, 66, 67, 70, 86, 87, 88, and 90, further comprising the step of obtaining said recombinant protein from said host cell. 94. A method of producing an antibody specific for a recombinant protein, comprising: performing the method of any one of Claims 1, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 86, 88, and 90 to produce said recombinant protein; and crystallizing said recombinant protein.

95. A method for generating an antibody against a recombinant protein, comprising: performing the method of any one of Claims 1, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 86, 88, and 90 to produce said recombinant protein; and using said recombinant protein to immunize a subject.

96. A method of assaying a biological activity of a recombinant protein, comprising: performing the method of any one of Claims 1, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 86, 88, and 90 to produce said recombinant protein; and using said recombinant protein in a biological assay.