WO2006078273A2 - Methods and compositions for producing recombinant proteins - Google Patents

Abstract

This invention relates to methods and compositions for producing recombinant proteins, especially in high yield. The invention is particularly amenable to producing proteins that are insoluble or minimally soluble in aqueous buffers. In particular, the invention concerns the use of fusion molecules in which a desired protein is produced as a fusion molecule linked to solubility facilitating protein, such as a chaperone protein or the phage T7 protein kinase.

Description

Title of the Invention:

Methods and Compositions for Producing Recombinant Proteins

Field of the Invention: [0001] This invention relates to methods and compositions for producing recombinant proteins, especially in high yield. The invention is particularly amenable to producing proteins that are insoluble or minimally soluble in aqueous buffers. In particular, the invention concerns the use of fusion molecules in which a desired protein is produced as a fusion molecule linked to a solubility facilitating protein.

Cross-Reference to Related Applications

[0002J This application claims priority to U.S. Patent Application Serial No. 60/564,982, filed April 26, 2004, which application is herein incorporated by reference in its entirety.

Background of the Invention:

A. Recombinant Expression of Proteins

|0003] Recombinant DNA methods (U.S. Patent No. 4,237,224; Sambrook, J., et al,. In: Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (2001); Glick, B.R. et al, In: Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, DC (2003); Watson, J.D. et al., In: Recombinant DNA, W. H. Freeman Company, New York (1992)) using the bacterium Escherichia coli have been widely successful at producing desired proteins (e.g., vaccines, antibodies, enzymes, hormones, etc.) in amounts that have permitted their significant commercial and scientific exploitation.

[0004] Although other expression systems exist, the E. coli expression system offers many advantages. It is easy to handle, cost-effective, and can produce proteins in high yield (Schmidt, M. et al. (2002) "EXPRESSION SYSTEMS FOR PRODUCTION OF RECOMBINANT ALLERGENS," Int Arch Allergy Immunol. 128(4):264-270). Unfortunately, however, such enhanced production is frequently accompanied by problems of protein insolubility, production host non-viability, and aberrant protein folding (Zhang, Z. et al. (2003) "PRODUCTION OF SOLUBLE AND FUNCTIONAL ENGINEERED ANTIBODIES IN ESCHERICHIA COLI IMPROVED BY FKPA," Biotechniques. 35(5):1032-8, 1041-1042; Zhang, Z. et al. (2002) "[ESCHERICHIA COLI DISULFIDE-FORMING RELATED PROTEINS: STRUCTURES, FUNCTIONS AND THEIR APPLICATION IN GENE ENGINEERING FOR EXPRESSING HETEROLOGOUS PROTEINS IN ESCHERICHIA COLI] (ENGLISH TRANSLATION)" Sheng Wu Gong Cheng Xue Bao. 18(3):261-266; Makrides, S.C. (1996) "STRATEGIES FOR ACHIEVING HIGH-LEVEL EXPRESSION OF GENES IN ESCHERICHIA COLI," Microbiol. Rev. 60:512-538; Lund, P.A. (2001) "MICROBIAL MOLECULAR CHAPERONES," Adv Microb Physiol. 44:93-140; Georgiou, G. et al. "EXPRESSION OF CORRECTLY FOLDED PROTEINS IN ESCHERICHIA COLI," Curr Opin Biotechnol. 7(2): 190- 197).

[0005] Many strategies have been proposed to address these problems. These include modifying expression conditions (e.g., medium composition, temperature and induction parameters), employing specialized promoter systems (e.g., tet, T7, tac, ara, etc.), employing specialized host strains, etc. (Ashraf, S. S. el al. (2004) "A NOVEL MULTI-AFFINITY TAG SYSTEM TO PRODUCE HIGH LEVELS OF SOLUBLE AND BIOTINYLATED PROTEINS IN ESCHERICHIA COLI," Protein Expr Purif. 33(2):238-245; Georgiou, G. et al. "EXPRESSION OF CORRECTLY FOLDED PROTEINS IN ESCHERICHIA COLI," Curr Opin Biotechnol. 7(2):190-197; Makrides, S.C. (1996) "STRATEGIES FOR ACHIEVING HIGH-LEVEL EXPRESSION OF GENES IN ESCHERICHIA COLl," Microbiol. Rev. 60:512-538; Hockney, R.C. (1994) "RECENT DEVELOPMENTS IN HETEROLOGOUS PROTEIN PRODUCTION IN ESCHERICHIA COLI," Trends Biotechnol. 12(1 1):456-463; Buchner, J. et al. (1991) "ROUTES To ACTIVE PROTEINS FROM TRANSFORMED MICROORGANISMS," Curr Opin Biotechnol. 2(4):532-538; Lilie, H. et al. (1998) "ADVANCES IN REFOLDING OF PROTEINS

PRODUCED IN E. COLI," Curr Opin Biotechnol. 9(5):497-501 ; Panda, A.K. (2003) "BlOPROCESSING OF THERAPEUTIC PROTEINS FROM THE INCLUSION BODIES OF

ESCHERICHIA COLI," Adv Biochem Eng Biotechnol. 85:43-93; Sandkvist, M. Bagdasarian M. (1996) "SECRETION OF RECOMBINANT PROTEINS BY GRAM- NEGATIVE BACTERIA," Curr Opin Biotechnol. 7(5):505-51 1 ; Danese, P.N. et al. ( 1998) "TARGETING AND ASSEMBLY OF PERIPLASMS AND OUTER-MEMBRANE PROTEINS IN ESCHERICHIA COLI," Annu Rev Genet. 32:59-94; Weickert, M.J. et al. (1996) "OPTIMIZATION OF HETEROLOGOUS PROTEIN PRODUCTION IN ESCHERICHIA COLI," Curr Opin Biotechnol. 7(5):494-499; Blight, M.A. et al. (1994) "PROTEIN SECRETION PATHWAY IN ESCHERICHIA COLI," Curr Opin Biotechnol. 5(5):468-474; Georgiou, G. et al. (1996) "EXPRESSION OF CORRECTLY FOLDED PROTEINS IN ESCHERICHIA COLI," Curr Opin Biotechnol. 1996 Apr;7(2):190-197; Swartz, J.R.

(2001) "ADVANCES IN ESCHERICHIA COLI PRODUCTION OF THERAPEUTIC PROTEINS," Curr Opin Biotechnol. 12(2):195-201; Andersen, D.C. et al. (2002) "RECOMBINANT PROTEIN EXPRESSION FOR THERAPEUTIC APPLICATIONS," Curr Opin Biotechnol. 13(2): 117-123; Hitt, M.M. et al. (2000) "Gene Vectors For

Cytokine Expression In Vivo," Curr Pharm Des. 6(6):613-632; Schmidt, M. et al.

(2002) "EXPRESSION SYSTEMS FOR PRODUCTION OF RECOMBINANT ALLERGENS," Int Arch Allergy Immunol. 128(4):264-270; Zhang, Z. et al. (2003) "PRODUCTION OF SOLUBLE AND FUNCTIONAL ENGINEERED ANTIBODIES IN ESCHERICHIA COLI IMPROVED BY FKPA," Biotechniques. 35(5): 1032-8, 1041-2; Zhang, Z. et al. (2002) "[ESCHERICHIA COLI DISULFIDE-FORMING RELATED PROTEINS: STRUCTURES, FUNCTIONS AND THEIR APPLICATION IN GENE ENGINEERING FOR EXPRESSING HETEROLOGOUS PROTEINS IN ESCHERICHIA COLI] (ENGLISH TRANSLATION)" Sheng Wu Gong Cheng Xue Bao. 18(3):261-266).

[0006| In particular, the problem of insolubility has been addressed through the use of fusion vectors that mediate the expression of a fusion product composed of the desired target gene linked to a peptide signal sequence or to a "chaperone" or "carrier" protein that is capable of "escorting" the fusion protein out of the cytoplasm and into the periplasmic space (see, Sheibani, N. (1999) "Prokaryotic gene fusion expression systems and their use in structural and functional studies of proteins," Prep Biochem Biotechnol. 1999 Feb;29(l):77-90; Lund, P.A. (2001) "MICROBIAL MOLECULAR CHAPERONES," Adv Microb Physiol. 44:93-140). Chaperone proteins are discussed by Ashraf, S. S. et al. (2004) "A NOVEL M U LTI- AFFINITY TAG SYSTEM TO PRODUCE HIGH LEVELS OF SOLUBLE AND BlOTlNYLATED PROTEINS IN ESCHERICHIA COLl," Protein Expr Purif. 33(2):238- 245; Bach, H. et al. (2001) "ESCHERICHIA COLi MALTOSE-BINDING PROTEIN AS A MOLECULAR CHAPERONE FOR RECOMBINANT INTRACELLULAR CYTOPLASMIC SINGLE-CHAIN ANTIBODIES," J MOI Biol. 312(l):79-93; Bulieris, P.V. et al. (2003) "FOLDING AND INSERTION OF THE OUTER MEMBRANE PROTEIN OMPA IS ASSISTED BY THE CHAPERONE SKP AND BY LIPOPOLYSACCHARIDE," J Biol Chem. 278(11):9092-9099; Chen, J. et al. (1999) "CHAPERONE ACTIVITY OF DSBC," J Biol Chem. 274(28):19601-19605; Fox, J.D. et al. (2003) "MALTODEXTRIN- BiNDiNG PROTEINS FROM DIVERSE BACTERIA AND ARCHAEA ARE POTENT SOLUBILITY ENHANCERS," FEBS Lett. 537(l-3):53-57; Guan, M. et al. (2002) "PRODUCTION OF EXTRACELLULAR DOMAIN OF HUMAN TISSUE FACTOR USING MALTOSE-BINDING PROTEIN FUSION SYSTEM," Protein Expr Purif. 26(2):229-234; Hayhurst, A. et al. (1999) "ESCHERICHIA COLI SKP CHAPERONE COEXPRESSION IMPROVES SOLUBILITY AND PHAGE DISPLAY OF SINGLE-CHAIN ANTIBODY FRAGMENTS," Protein Expr Purif. 15(3):336-343; Kapust, R.B. et al. ( 1999) "ESCHERICHIA COLI MALTOSE-BINDING PROTEIN IS UNCOMMONLY EFFECTIVE AT PROMOTING THE SOLUBILITY OF POLYPEPTIDES TO WHICH IT IS FUSED," Protein Sci. 8(8): 1668-1674; Liu, X. et al. (2001 ) "DISULFIDE-DEPENDENT FOLDING AND EXPORT OF ESCHERICHIA COLI DSBC," J Biol Chem. 276(2): 1 146-1 151 ; Shrestha,

A. et al. (2004) "BACTERIAL CHAPERONE PROTEIN, SKP, INDUCES LEUKOCYTE

CHEMOTAXIS VIA C5A RECEPTOR," Am J Pathol. 164(3):763-772; Yin, G. et al. (2004) "ENHANCING MULTIPLE DISULFIDE BONDED PROTEIN FOLDING IN A CELL- FREE SYSTEM," Biotechnol Bioeng. 86(2): 188- 195; and Yokoyama, S. (2003) "PROTEIN EXPRESSION SYSTEMS FOR STRUCTURAL GENOMICS AND PROTEOMICS," Curr Opin Chem Biol. 7(l):39-43).

[0007 j In some embodiments of this approach, additional "affinity tag" peptide sequences (such as HiS₆, thioredoxin, nusA, glutathione S-transferase (GST),

Strep-tag, FLAG ® (Sigma Aldrich), AviTag, calmodulin-binding peptide (CBP), etc.) are incorporated into the fusion protein in order to facilitate the eventual recovery and purification of the desired protein (Terpe, K. (2003) "OVERVIEW OF TAG PROTEIN FUSIONS: FROM MOLECULAR AND BIOCHEMICAL FUNDAMENTALS TO COMMERCIAL SYSTEMS," Appl Microbiol Biotechnol. 60(5):523-533; Alexandrov, A. et al. (2001) "MBP FUSION PROTEIN WITH A VIRAL PROTEASE CLEAVAGE SITE: ONE-STEP CLEAVAGE / PURIFICATION OF INSOLUBLE PROTEINS." Biotechniques. 30(6): 1194-1 198; Cattoli, F. et al. (2002) "SEPARATION OF MBP FUSION PROTEINS THROUGH AFFINITY MEMBRANES," Biotechnol Prog. 18(l):94-100; Ishii, Y. et al. (1998) "SINGLE-STEP PURIFICATION AND CHARACTERIZATION OF MBP (MALTOSE BINDING PROTEIN)-DNAJ FUSION PROTEIN AND ITS UTILIZATION FOR STRUCTURE- FUNCTION ANALYSIS," J Biochem (Tokyo). 124(4):842-847; Nomine, Y. et al. (2001) "FORMATION OF SOLUBLE INCLUSION BODIES BY HPV E6 ONCOPROTEIN FUSED To MALTOSE-BINDING PROTEIN," Protein Expr Purif. 23(l):22-32; Schafer, U. et al. (1999) "SKP, A MOLECULAR CHAPERONE OF GRAM-NEGATIVE BACTERIA, Is REQUIRED FOR THE FORMATION OF SOLUBLE PERIPLASMS INTERMEDIATES OF OUTER MEMBRANE PROTEINS," J Biol Chem. 274(35):24567-24574; Schierle, CF. et al. (2003) "THE DSBA SIGNAL SEQUENCE DIRECTS EFFICIENT,

COTRANSLATIONAL EXPORT OF PASSENGER PROTEINS TO THE ESCHERICHIA COLI

PERIPLASM VIA THE SIGNAL RECOGNITION PARTICLE PATHWAY," J Bacteriol. 185(19):5706-5713; Zhang, Q. et al. (2004) "EXPRESSION OF THE SOLUBLE EXTRACELLULAR DOMAIN OF HUMAN THROMBOPOIETIN RECEPTOR USING A MALTOSE-BINDING PROTEIN-AFFINITY FUSION SYSTEM," Biol Pharm Bull. 27(2):219-221 ; Zhang, Z. et al. (2002) "OVEREXPRESSION OF DSBC AND DSBG MARKEDLY IMPROVES SOLUBLE AND FUNCTIONAL EXPRESSION OF SINGLE-CHAIN Fv ANTIBODIES IN ESCHERICHIA COLI " Protein Expr Purif. 26(2):218-228; Zhang,

Z. et al. (2002) "[ESCHERICHIA COL/ DlSULFIDE-FORMING RELATED PROTEINS:

STRUCTURES, FUNCTIONS AND THEIR APPLICATION IN GENE ENGINEERING FOR EXPRESSING HETEROLOGOUS PROTEINS IN ESCHERICHIA COLI] (ENGLISH TRANSLATION)" Sheng Wu Gong Cheng Xue Bao. 18(3):261 -266; Levy, R. et al. (2001) "PRODUCTION OF CORRECTLY FOLDED FAB ANTIBODY FRAGMENT IN THE CYTOPLASM OF ESCHERICHIA COLI TRXB GOR MUTANTS VIA THE COEXPRESSION OF MOLECULAR CHAPERONES," Protein Expr Purif. 23(2):338-347). Such sequences include affinity "tags" such as FLAG, c-myc, S-tag, AviTag, and His₆ (Ashraf, S. S. et al. (2004) "A NOVEL MULTI-AFFINITY TAG SYSTEM TO PRODUCE HIGH LEVELS OF SOLUBLE AND BIOTINYLATED PROTEINS IN ESCHERICHIA COLI," Protein Expr Purif. 33(2):238-245; Terpe, K. (2003) "OVERVIEW OF TAG PROTEIN FUSIONS: FROM MOLECULAR AND BIOCHEMICAL FUNDAMENTALS TO COMMERCIAL

SYSTEMS," Appl Microbiol Biotechnol. 60(5):523-533; Nilsson, J et al. (1996) "MULTIPLE AFFINITY DOMAINS FOR THE DETECTION, PURIFICATION AND IMMOBILIZATION OF RECOMBINANT PROTEINS," J. MOI. Recognit. 9(5-6):585-594).

[0008] Several gene constructs, comprising vectors for forming chaperone protein fusions with desired gene sequences are commercially available from Novagen (www.novagen.com) (e.g., pET-39b and 40b which employ Dsb tags for export and periplasmic folding of target proteins; pET-41a-c and 42a-c which employ GST fusion tags for enhanced production and solubility; pET-43.1 a-c which employ a 495 amino acid long NusA (Nus'Tag™) protein; pET-44a-c which contains a Nus'Tag™ sequence plus N- and C-terminal His'Tag sequences, pET- 45b(+)which contains an amino-terminal His*Tag™ sequence and minimal extraneous sequences; pET-46 Ek/LIC prepared vector which contains an amino- terminal His^#Tag™ sequence).

[0009] For example, overexpression of a DsbC-single chain Fv antibody fusion protein led to enhanced production and solubility of the single chain Fv antibody Zhang, Z. et al. (2002) "OVEREXPRESSION OF DSBC AND DSBG MARKEDLY IMPROVES SOLUBLE AND FUNCTIONAL EXPRESSION OF SINGLE-CHAIN FV ANTIBODIES IN ESCHERICHIA COLI " Protein Expr Purif. 26(2):218-228; Zhang, Z. et al. (2003) "PRODUCTION OF SOLUBLE AND FUNCTIONAL ENGINEERED ANTIBODIES IN ESCHERICHIA COLI IMPROVED BY FKPA," Biotechniques. 35(5): 1032-8, 1041- 1042; Zhang, Z. et al. (2002) "[ESCHERICHIA COLI DlSULFIDE-FORMING RELATED

PROTEINS: STRUCTURES, FUNCTIONS AND THEIR APPLICATION IN GENE ENGINEERING FOR EXPRESSING HETEROLOGOUS PROTEINS IN ESCHERICHIA COLI] (ENGLISH TRANSLATION)" Sheng Wu Gong Cheng Xue Bao. 18(3):261-266). Significantly, the chaperone fusions used by these researchers contained a signal sequence that directs the fusion protein to the periplasmic space. Such expression has been found to be problematic. Some proteins have difficulty traversing the cytoplasmic membrane, can aggregate in the periplasm, and can contribute to cell toxicity issues. To address these problems, Levy, R. el al. (2001) ("PRODUCTION OF CORRECTLY FOLDED FAB ANTIBODY FRAGMENT IN THE CYTOPLASM OF ESCHERICHIA COLI TRXB GOR MUTANTS VIA THE COEXPRESSION OF MOLECULAR CHAPERONES," Protein Expr Purif. 23(2):338-347) disclose the co-expression of a target gene (i.e., a single chain antibody Fv fragment) and a chaperone protein construct in which the signal sequence of the chaperone protein had been deleted so that the expressed proteins were retained in the cytoplasm. However, since the E. coli cytoplasm is normally maintained at a low redox potential, and as a result, oxidative protein folding (such as that involved in disulfide bond formation) does not normally occur there. Thus, in order to permit proper folding of expressed proteins in the cytoplasm, the use of specialized (trxB^~ gor^') host cells is taught (Levy, R. et al. (2001) "PRODUCTION OF CORRECTLY FOLDED FAB ANTIBODY FRAGMENT IN THE CYTOPLASM OF ESCHERICHIA COLI TRXB GOR MUTANTS VIA THE COEXPRESSION OF MOLECULAR CHAPERONES," Protein Expr Purif. 23(2):338- 347).

[0010] Unfortunately, despite such advances, an enhanced ability to produce soluble proteins remains often accompanied by an attenuation in protein yield. Thus, a need remains for methods and compositions that provide soluble proteins that are correctly folded and in functional form without unacceptably diminishing the yield of recovered protein or requiring complex host strains. The present invention, which permits high yield production (and in some embodiments, also recovery) of properly folded and functional proteins, is directed to these and other needs.

Brief Description of the Figures:

[00111 Figure 1 shows the expression of total (T) and soluble (S) proteins Wnt5A, Folliculin, and IFN-Hyb3 using a Skp fusion expression system.

[0012| Figure 2 shows the expression of total (T) and soluble (S) proteins YopD and Endostatin using a Skp fusion expression system. [0013J Figure 3 shows the expression of total (T) and soluble (S) proteins Hifl A, IL-13, a domain of Folliculin (FD) and full-length Folliculin (F) using a Skp fusion expression system.

[0014J Figure 4 shows the expression of total (T) and soluble (S) proteins Folliculin, IFN-Hyb3, Wnt5a, YopD and Endostatin using a MBP fusion expression system.

[0015] Figure 5 shows the expression of total (T) and soluble (S) proteins Folliculin, IFN-Hyb3, and Wnt5a using a DsbC fusion expression system.

[0016] Figure 6 illustrates the positions of the genetic determinants of Skp fusion plasmid pDest 579, and the DsbC fusion plasmid pDest-568.

[0017] Figure 7 demonstrates that Folliculin is highly insoluble when expressed in E. coli even in the presence of various chaperones, GroEL- ES/Trigger factor (TF) or Skp/DsbC. Arrows indicate the position of the chaperones or the fusion proteins. T = Total protein; S = Soluble protein; M = Marker.

[0018] Figure 8 illustrates the configuration of preferred fusion proteins of the Protein Kinase Protein (PK) and a Target Protein. AF = Affinity Tag.

[0019] Figure 9 illustrates the ability of the PK fusion protein to facilitate the solubility of Hifl a, Folliculin, a Folliculin domain (FD) and ILl 3. An amino terminal PK fusion is used. Arrows indicate the position of fusion proteins. T = Total protein; S = Soluble protein. All proteins shown were almost completely insoluble with an N-HiS₆ affinity tag.

[0020] Figure 10 shows the purification of Strep Il-PK-Folliculin using Strep- tactin. L = Load; F = Flow through; W = Wash; 1 -6 represent elution fractions. Arrows between 2 and 3 indicate possible proteolytic fragments; the position of Folliculin is identified on the right.

[00211 Figure 11 shows the purification of PK-DEFG Folliculin using Strep- tactin. L = Load; F = Flow through; W = Wash; 1-6 represent elution fractions. Arrows between 1 and 2 indicate possible proteolytic fragments; the position of PK-DEFG is identified on the right.

[0022] Figure 12 shows TEV protease cleavage of Folliculin and Folliculin domain fusion.

[0023] Figure 13 shows a comparison of the level of expression and solubility of Folliculin obtained with StrepII-Skp or Strepll-PK fusion partners with Tev5 and TevlO Fusions. T = Total protein; S = Soluble protein; P = Pellet.

[0024] Figure 14 shows the purification of Strep-tag II-PK-Folliculin TevlO fusion protein.

[0025] Figure 15 shows TEV protease digestion of a Strepll-PK-TEV 10- Folliculin fusion molecule.

Summary of the Invention:

[0026] The production of large quantities of soluble and correctly folded proteins is essential for applications such as structure determination, functional analysis, and clinical or therapeutic trials. However, the use of recombinant methods to obtain proteins is limited by two problems. First, the heterologous expression of recombinant proteins often leads to the formation of insoluble aggregates. Second, most proteins when expressed in heterologous hosts are often produced in very low amounts. Although the yield of a target protein may be improved by fusing it to a fusion partner, common fusion partners do not solve the solubility problems. The present invention offers solutions to both of these problems.

[0027] The present invention thus relates to recombinant methods and compositions for producing recombinant proteins, especially in high yield. The invention is particularly amenable to producing proteins that are typically insoluble or minimally soluble in aqueous buffers when produced by other recombinant methods. In particular, the invention concerns the use of fusion molecules in which a desired protein is produced as a fusion molecule linked to a chaperone protein, to a phage T7 protein kinase, or to a fragment thereof. [0028] Recombinant proteins and their fragments are produced in E. coli or other hosts for a variety of reasons such as basic scientific research, structure determinations, clinical or therapeutic applications, etc. However, the applicability of recombinant expression procedures is often limited by the difficulty of producing proteins at sufficiently high yields. Even where high yields are attained, the expressed protein is frequently produced in insoluble and/or non-functional forms. Unfortunately, existing expression "tag" systems do not fully address these deficiencies.

[0029] The present invention offers a solution to these problems. In accordance with preferred embodiments of the present invention solubility facilitating proteins are fused to a desired target protein (to form an "expression tag") and employed to facilitate the expression of the target protein. In one embodiment, such solubility facilitating proteins may comprise a chaperone protein. Chaperone proteins are expressible in high yield, and help to make proteins fold correctly. Alternatively, the solubility facilitating proteins of the present invention may comprise the phage T7 protein kinase, or a fragment thereof. In a particularly preferred embodiment of the present invention polynucleotides encoding such solubility facilitating proteins are fused to the N- or C-terminus of a polynucleotide encoding a desired target protein (either directly, or through a linking sequence). Preferably, when a chaperone protein is employed as the solubility facilitating protein, such polynucleotides will not encode the chaperone protein's signal sequence. As a consequence, the expressed proteins are retained in the cytoplasm rather than the periplasm. Such cytoplasmic retention prevents possible interference with cell viability that might occur if the expressed proteins were secreted into the periplasm. Although any of a variety of suitable chaperone proteins may be employed in concert with the present invention, the use of the signal sequence-less chaperone proteins: Skp, DsbC in such "Expression Tags" is particularly preferred. The use of additional sequences (e.g., poly-His, Strep-tag or Maltose Binding Protein (MBP) are preferred.

|0030| In detail, the invention provides a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises a solubility facilitating protein, (especially a chaperone protein, or a phage T7 protein kinase, or a fragment thereof), linked to a desired target protein.

|0031] The invention particularly concerns the embodiment of such a nucleic acid molecule wherein the solubility facilitating protein comprises a fragment of the chaperone protein or the phage T7 protein kinase. The invention further concerns the embodiments of such nucleic acid molecules wherein the encoded fragment of the chaperone protein lacks a signal sequence.

[0032] The invention concerns the embodiments of such nucleic acid molecules wherein the chaperone protein is selected from the group consisting of E. coli DsbC protein and E. coli Skp protein.

[0033] The invention concerns the embodiments of such nucleic acid molecules wherein the solubility facilitating protein is the phage T7 protein kinase, or a fragment thereof, and/or wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).

[0034] The invention concerns the embodiments of such nucleic acid molecules wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).

[0035J The invention further concerns a method of producing a desired target protein, wherein the method comprises the steps:

(A) introducing a plasmid vector capable of being expressed in a host cell into the host cell, wherein the vector comprises a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises a solubility facilitating protein (especially a chaperone protein, a phage T7 protein kinase, or a fragment of either), linked to the desired target protein;

(B) permitting the host cell to express the fusion protein;

(C) recovering the fusion protein from the cytoplasm of the host cell; and (D) recovering the desired target protein from the fusion protein.

|0036| The invention further concerns the embodiments of such method wherein the solubility facilitating protein comprises a fragment of a chaperone protein, and/or wherein such fragment lacks a signal sequence, and/or wherein the chaperone protein is selected from the group consisting of E. coli DsbC protein and E coli Skp protein, and/or wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).

[0037] The invention further concerns the embodiments of such method wherein the solubility facilitating protein comprises a fragment of the phage T7 protein kinase, or a fragment thereof, and/or wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).

[0038] The invention further concerns a desired target protein produced by the process of:

(A) introducing a plasmid vector capable of being expressed in a host cell into the host cell, wherein the vector comprises a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises a solubility facilitating protein (especially, a chaperone protein, a phage T7 protein kinase, or a fragment of either), linked to the desired target protein;

(B) permitting the host cell to express the fusion protein;

(C) recovering the fusion protein from the cytoplasm of the host cell; and (D) obtaining the desired target protein from the fusion protein.

[0039] The invention further concerns the embodiments of such method wherein the solubility facilitating protein comprises a fragment of a chaperone protein, and/or wherein such fragment lacks a protein signal sequence, and/or wherein the chaperone protein is selected from the group consisting of E. coli DsbC protein and E. coli Skp protein, and/or wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).

[0040J The invention further concerns the embodiments of such method wherein the fusion protein encodes a phage T7 protein kinase, or a fragment thereof, and/or wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).

Description of the Preferred Embodiments: [0041] Production of recombinant proteins has been an important method for production of medically relevant proteins and developing new therapeutic molecules. However, expression using heterologous target genes has production problems, especially insolubility of the expressed proteins (Rudolph, R. et al. (1996) "IN VITRO FOLDING OF INCLUSION BODY PROTEINS," FASEB J. 10:49-56). Even though advancement has been made in recent years for efficient protein production, most of the target proteins cannot be properly folded manner and usually end in inclusion bodies (Christendat, D. et al. (2000) "STRUCTURAL PROTEOMICS: PROSPECTS FOR HIGH THROUGHPUT SAMPLE PREPARATION," Prog. Biophys. MoI. Biol. 73:339-345). Many approaches including optimization of expression condition have been tried to obtain correctly folded active protein (Zhang, Y. et al. (1998) "EXPRESSION OF EUKARYOTIC PROTEINS IN SOLUBLE FORM IN ESCHERICHIA COLI," Protein Expr. Purif. 12:159-165; Hwang, H. S. et al. (2002) "PREPARATION OF ACTIVE RECOMBINANT CATHEPSIN K EXPRESSED IN BACTERIA AS INCLUSION BODY," Protein Expr. Purif. 25:541-546; Thomas, J.G. et AL. ( 1997) "DIVERGENT EFFECTS OF CH APERONE OVEREXPRESSION AND ETH ANOL SUPPLEMENTATION ON INCLUSION BODY FORMATION IN RECOMBINANT ESCHERICHIA COLI," Protein Expr. Purif. 1 1 :289-296). Unfortunately, the methods have been mostly target-specific and not amenable for general application. In addition, the productive use of mRNA, particularly when produced under a strong T7 promoter, becomes an issue because all mRNAs do not get used to make proteins and is unstable (Lopez, P.J. et al. (1994) "THE USE OF TRNA AS A TRANSCRIPTION REPORTER: THE T7 LATE PROMOTER IS EXTREMELY EFFICIENT IN E. COLi BUT ITS TRANSCRIPTS ARE POORLY EXPRESSED," Nucleic Acids Res. 22:1 186-1 193; lost, \. et al. {\ 995) "THE STABILITY OF E. COLI LACZ MRN A DEPENDS UPON THE SIMULTANEITY OF ITS SYNTHESIS AND TRANSLATION," EMBO J 14:3252-3261). To address these issues, the present invention provides a "tag" that addresses both solubility and mRNA stability problems. As a consequence, significant amounts of soluble proteins can be recovered.

[0042] The present invention relates to recombinant methods and compositions for producing recombinant proteins, especially in high yield. The invention is particularly amenable to producing proteins that are typically insoluble or minimally soluble (i.e., where less than 50% and more preferably if less than 30%, still more preferably less than 20%, and most preferably less than 10% of total protein produced is insoluble) in aqueous buffers when produced using other recombinant methods. In particular, the invention concerns the use of fusion molecules in which a desired protein is produced as a fusion molecule linked to a solubility facilitating protein, especially a chaperone protein or the phage T7 protein kinase. The invention particularly pertains to the use of the E. coli DsbC protein, the E. coli Skp protein, or a protein derived from such proteins (e.g., a truncated form of such proteins, etc.) as chaperone proteins (e.g., fusion partners) to mediate the enhanced expression of the target proteins, and to the use of the phage T7 protein kinase, or a protein derived therefrom (e.g., a truncated form of the phage T7 protein kinase, etc.) as a fusion partner to mediate the enhanced expression of the target proteins. In preferred embodiments, fusion molecules of such proteins are used in conjunction with affinity tags (e.g., proteins or polypeptides that can serve to facilitate the recovery or purification of the expressed fusion protein).

Methods And Compositions Of The Invention

|0043] The present invention relates to the use of gene fusions to improve the yield and to address the insolubility of desired target proteins produced through the use of recombinant DNA techniques. As used herein, a "target" protein is any recombinant protein whose production and/or recovery is desired. Target proteins may comprise any protein including enzymes, hormones, cytokines, growth factors, etc. Such proteins may be of prokaryotic origin (e.g., bacterial, viral, etc.) or eukaryotic (e.g., fungal, mammalian (especially human), etc.). The present invention particularly pertains to the use of Escherichia coli as a host cell for protein production. As will be appreciated, however, the principles of the present invention may be readily adapted to permit the use of the present invention in other host cells.

[0044J In accordance with the principles of the present invention, fusion polynucleotides encoding the target protein are operably linked to polynucleotides encoding one or more solubility facilitating proteins, or one or more functional domains thereof, are employed. Such fusion polynucleotides possess codons encoding the solubility facilitating protein (or domain(s) thereof) in frame with those encoding the target protein, so that, upon expression in a host cell, the fusion polynucleotide will mediate the production of a fusion protein comprising the solubility facilitating protein (or domain(s) thereof) and the target protein. As used herein, the term "solubility facilitating protein" is intended to refer to a protein that enhances the solubility of a desired target protein to which it is fused. Preferred solubility facilitating proteins include the phage T7 protein kinase and chaperone proteins. Suitable chaperone proteins are disclosed by Lund, P.A. (2001 ) "MICROBIAL MOLECULAR CHAPERONES," Adv Microb Physiol. 44:93-140; Bardwell, J.C. et al. (1993) "THE BONDS THAT TIE: CATALYZED DISULFIDE BOND FORMATION," Cell 74(5):769-771 ; Ellis, R.J. (1997) "MOLECULAR CHAPERONES: AVOIDING THE CROWD," Curr Biol. 7(9):R531 -R533; Ashraf, S.S. et al. (2004) "A NOVEL MULTI-AFFINITY TAG SYSTEM TO PRODUCE HIGH LEVELS OF SOLUBLE AND BIOTINYLATED PROTEINS IN ESCHERICHIA COLI," Protein Expr Purif. 33(2):238-245; Bach, H. et al. (2001) "ESCHERICHIA COLI MALTOSE-BINDING PROTEIN AS A MOLECULAR CHAPERONE FOR RECOMBINANT INTRACELLULAR CYTOPLASMIC SINGLE-CHAIN ANTIBODIES," J MOI Biol. 312(l):79-93; Bulieris, P.V. et al. (2003) "FOLDING AND INSERTION OF THE OUTER MEMBRANE PROTEIN OMPA IS ASSISTED BY THE CHAPERONE SKP AND BY LIPOPOLYSACCHARIDE," J Biol Chem. 278(1 1):9092-9099; Chen, J. et al. (1999) "CH APERONE ACTIVITY OF DSBC," J Biol Chem. 274(28): 19601 -19605; Fox, J. D. et al. (2003) "MALTODEXTRIN-BINDING PROTEINS FROM DIVERSE BACTERIA AND ARCHAEA ARE POTENT SOLUBILITY ENHANCERS," FEBS Lett. 537(1 -3):53-57; Guan, M. et al. (2002) "PRODUCTION OF EXTRACELLULAR DOMAIN OF HUMAN TISSUE FACTOR USING MALTOSE-BINDING PROTEIN FUSION SYSTEM," Protein Expr Purif. 26(2):229-234; Hayhurst, A. et al. (1999) "ESCHERICHIA COLI SKP CHAPERONE

COEXPRESSION IMPROVES SOLUBILITY AND PHAGE DISPLAY OF SINGLE-CHAIN ANTIBODY FRAGMENTS," Protein Expr Purif. 15(3):336-343; Kapust, R.B. et al. (1999) "ESCHERICHIA COLI MALTOSE-BINDING PROTEIN IS UNCOMMONLY EFFECTIVE AT PROMOTING THE SOLUBILITY OF POLYPEPTIDES TO WHICH IT IS FUSED," Protein Sci. 8(8):1668-1674; Liu, X. et al. (2001) "DISULFIDE-DEPENDENT FOLDING AND EXPORT OF ESCHERICHIA COLI DSBC," J Biol Chem. 276(2): 1 146- 1 151 ; Shrestha, A. et al. (2004) "BACTERIAL CHAPERONE PROTEIN, SKP, INDUCES LEUKOCYTE CHEMOTAXIS VIA C5A RECEPTOR," Am J Pathol. 164(3):763-772; Yin, G. et al. (2004) "ENHANCING MULTIPLE DISULFIDE BONDED PROTEIN FOLDING IN A CELL-FREE SYSTEM," Biotechnol Bioeng. 86(2): 188-195; and Yokoyama, S. (2003) "PROTEIN EXPRESSION SYSTEMS FOR STRUCTURAL GENOMICS AND PROTEOMICS," Curr Opin Chem Biol. 7(l):39-43.

[0045] The invention particularly concerns the embodiments in which the solubility facilitating protein domain(s) are selected from the group consisting of heat shock proteins (HsP), DsbC, Skp or MBP chaperone proteins, or the phage T7 protein kinase, or proteins, or domains thereof, derived therefrom. In particular, the use of a fusion partner derived from a phage T7 protein called "protein kinase" is preferred. This protein has been shown to stimulate its own expression, and the stimulation occurs at both translational as well post-transcriptional levels. The level of expression of this protein itself is extremely high as it synthesizes up to 40% of the total E .coli protein even from a single copy gene. It is also an extremely soluble protein. The properties of the phage T7 protein kinase are reviewed by Marchand, I. et al. (2001 a) ["BACTERIOPHAGE T7 PROTEIN KINASE PHOSPHORYLATES RNASE E AND STABILIZES MRNAS SYNTHESIZED BY T7 RNA POLYMERASE," MOI Microbiol. 42(3):767-776], Marchand, 1. et al. (2001b) ["HIGH-LEVEL AUTOENHANCED EXPRESSION OF A SINGLE-COPY GENE IN ESCHERICHIA COLI: OVERPRODUCTION OF BACTERIOPHAGE T7 PROTEIN KINASE DIRECTED BY T7 LATE GENETIC ELEMENTS," Gene. 262(1 -2):231 -238] and Pai, S.H. et al. (1975) ["PROTEIN KINASE OF BACTERIOPHAGE T7. 2. PROPERTIES, ENZYME SYNTHESIS IN VITRO AND REGULATION OF ENZYME SYNTHESIS AND ACTIVITY IN VIVO," Eur J Biochem. 55(l):305-314].

|0046] The polynucleotide encoding the solubility facilitating protein domain(s) may be fused to the polynucleotide encoding the target protein in any of several orientations. For example, the polynucleotide encoding a chaperone protein or the T7 protein kinase, or a domain thereof, may be linked to that which encodes either the amino or carboxyl terminus of the target protein. Multiple chaperone proteins, T7 protein kinases, or domains thereof (which may be the same or different from one another, or combinations thereof) may be used in a single fusion molecule; likewise, multiple target molecules (which may comprise multiple copies of the same target molecule, or multiple different target molecules, or combinations thereof) may be used in a single fusion molecule.

[0047] Certain chaperone proteins possess signal sequences that serve to direct the expressed protein out of the cytoplasm and into the periplasm of a host cell. In some embodiments of the present invention, where such transport is desired, the fusion polynucleotide employed will contain the signal sequence(s) that are sufficient to mediate such transport. In more preferred embodiments, the fusion polynucleotide will encode only a fragment of the chaperone protein, and will not contain the chaperone protein's signal sequence. In this embodiment, the expressed protein will accumulate in the cytoplasm. The use of this embodiment is preferred for enhancing the production and/or recovery of proteins whose accumulation in the periplasm might affect cellular viability.

[0048] In a further preferred embodiment, the fusion polynucleotides of the invention may contain one or more additional polynucleotides in addition to those encoding the solubility facilitating protein or protein domain(s) and the target protein. In particular, the present invention contemplates that such fusion polynucleotides may contain polynucleotides that, upon expression, encode proteins or polypeptides that can serve to facilitate the recovery or purification of the expressed fusion protein. For example, such fusion polynucleotides may encode a poly-His peptide (e.g., HiS₆), poly-Arg peptide (e.g., Arg₆), streptavidin binding protein (SBP), nusA, TrxA, DsbA, calmodulin-binding peptide (CBP), calmodulin-binding domain (CBD), glutathione S-transferase (GST), FLAG ® (Sigma Aldrich), AviTag, chitin binding domain, etc. (see, Terpe, K. (2003) "OVERVIEW OF TAG PROTEIN FUSIONS: FROM MOLECULAR AND BIOCHEMICAL FUNDAMENTALS TO COMMERCIAL SYSTEMS," Appl Microbiol Biotechnol. 60(5):523-533). Such additional polynucleotide(s) may be placed before or after the solubility facilitating protein-encoding sequences, and before or after the polynucleotide sequences that encode the desired target protein.

Uses Of The Invention

|0049] The methods and compositions of the present invention are useful for producing catalytic proteins, such as enzymes, co-factors, etc. that may be used to catalyze chemical or biochemical reactions. Such proteins include restriction endonucleases, polymerases, exonucleases, proteases, peptidases, amylases, xylanases, cellulases, chitinases, lipases, hydrolases, hydrogenases, dehydrogenases, etc. (see, Haki, G.D. et al. (2003) "DEVELOPMENTS IN INDUSTRIALLY IMPORTANT THERMOSTABLE ENZYMES: A REVIEW," Bioresour Technol. 89(1): 17-34; Panke, S. et al. (2002) "ENZYME TECHNOLOGY AND BiOPROCESS ENGINEERING," Curr Opin Biotechnol. 13(2):1 1 1-1 16; Zhao, H. et al. (2002) "Directed evolution of enzymes and pathways for industrial biocatalysis," Curr Opin Biotechnol. 13(2): 104-1 10; Demain, A.L. (2001) "MOLECULAR GENETICS AND INDUSTRIAL MICROBIOLOGY~30 YEARS OF MARRIAGE," J Ind Microbiol Biotechnol. 2001 Dec;27(6):352-356; Zaks, A. (2001) "INDUSTRIAL BIOCATALYSIS," Curr Opin Chem Biol. 5(2):130-136; Schmid, A. et al. (2001) "INDUSTRIAL BIOCATALYSIS TODAY AND TOMORROW," Nature. 409(6817):258- 268). [0050] The methods and compositions of the present invention are further useful for producing diagnostic proteins, such as antigens, haptens, single chain antibodies, etc. (see, Blazek, D. et al. (2003) "THE PRODUCTION AND APPLICATION OF SINGLE-CHAIN ANTIBODY FRAGMENTS," Folia Microbiol (Praha).48(5):687- 698; Bilbao, G. et al. (2002) "Genetically engineered intracellular single-chain antibodies in gene therapy," MoI Biotechnol. 22(2): 191 -21 1 ; Arnon, R. et al. (2003) "OLD AND NEW VACCINE APPROACHES," Int Immunopharmacol. 3(8): 1 195- 204; Schmitt, J. et al. (2002) "RECOMBINANT AUTOANTIGENS," Autoimmun Rev. l (l-2):79-88; Liljeqvist, S. et al. (1999) "Production of recombinant subunit vaccines: protein immunogens, live delivery systems and nucleic acid vaccines," J Biotechnol. 73(1): 1-33). Thus, the methods and compositions of the present invention may be used to produce diagnostic compositions useful in the diagnosis of diseases such as cancer, Alzheimer's disease, Parkinson disease, diabetes, inflammatory and autoimmune diseases, anemia, AIDS, SARS, influenza, etc.

[0051] The methods and compositions of the present invention are additionally useful for producing therapeutic proteins, such as humanized antibodies, albumins, hormones (e.g., insulin, growth hormone, etc.), receptors (e.g., adrenocorticotropic hormone receptor and its bioactive fragments, angiotensin receptor, atrial natriuretic receptor, bradykininin receptor, growth hormone receptor, chemotatic receptor, dynorphin receptor, endorphin receptor, the receptor for β-lipotropin and its bioactive fragments, enkephalin receptor, enzyme inhibitor receptors, the receptor for fibronectin and its bioactive fragments, gastrointestinal- and growth hormone-releasing peptide receptors, the receptor for luteinizing hormone releasing peptide, the receptor for melanocyte stimulating hormone, neurotensin receptor, opioid receptor, oxytocin receptor, vasopressin receptor, vasotocin receptor, the receptor for parathyroid hormone and fragments, protein kinase receptor, somatostatin receptor, substance P receptor), and ligands thereof, chemokines, cytokines, growth factors (e.g., transforming growth factor, nerve growth factor, brain derived growth factor, neurotrophin-3, neurotrophin-4, heptaocyte growth factor, T-cell Growth Factor (TGF, TGF-β 1 , TGF-β2, TGF-β3, etc.), Colony Stimulating Factors (G-CSF, GM-CSF, M-CSF etc.), tumor necrosis factor (TNF), Epidermal Growth Factor (EGF, LIF, KGF, OSM, PDGF, IGF-I, etc.), Fibroblast Growth Factor (αFGF, βFGF, etc.), interferons, blood factors, angiogenesis and erythropoiesis stimulators (e.g., erythropoietin (EPO)), colony stimulating factors, interleukins ("IL") (such as IL-1 , 1L-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, 1L-9, IL-I O, IL-1 1 , IL-12, IL-13, IL14, etc.); therapeutic vaccines, etc. (see, Renz, H. (1999) (1999) "SolubLE INTERLEUKIN-4 RECEPTOR (SIL-4R) IN ALLERGIC DISEASES," Inflamm Res. 48(8):425-431 ; Kobayashi, K. et al. (1998) "THE DEVELOPMENT OF RECOMBINANT HUMAN SERUM ALBUMIN," Ther Apher. 2(4):257-62; Arend, W.P. et al. (1998) "INTERLEUKIN-I RECEPTOR ANTAGONIST: ROLE IN BIOLOGY," Annu Rev Immunol. 16:27-55; Kiss, C. et al. (2004) "LEUKEMIC CELLS AND THE CYTOKINE PATCHWORK," Pediatr Blood Cancer. 42(2):1 13-121 ; Gitlitz, BJ. et al. (2003) "CYTOKINE-BASED THERAPY FOR METASTATIC RENAL CELL CANCER," Urol Clin North Am. 30(3):589-600; Murata, A. (2003) "Granulocyte colony-stimulating factor as the expecting sword for the treatment of severe sepsis," Curr Pharm Des. 9(14): 1 1 15-20; Ogata, H. et al.

(2003) "CYTOKINE AND ANTI-CYTOKINE THERAPIES FOR INFLAMMATORY BOWEL DISEASE," Curr Pharm Des. 9(14): 1 107-1 1 13; Davis, CB. et al. (2003) "IMMUNOCYTOKINES: AMPLIFICATION OF ANTI-C ANCER IMMUNITY," Cancer Immunol Immunother. 52(5):297-308; Rook, A.H. et al. (2002) " Cytokines and other biologic agents as immunotherapeutics for cutaneous T-cell lymphoma," Adv Dermatol. 18:29-43; Huffnagle GB, et al, "The Role Of Chemokines In Pneumonia," in Koch A, Strieter R, eds. CHEMOKINES IN DISEASE. Texas: R.G. Landis Co; 1996:151-168). Thus, the methods and compositions of the present invention may be used to produce pharmaceutical compositions useful in therapies for diseases such as cancer, Alzheimer's disease, Parkinson disease, diabetes, inflammatory and autoimmune diseases, anemia, AIDS, SARS, influenza, etc.

[0052] Having now generally described the invention, the same will be more readily understood through reference to the following examples and Figures, which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified. Example 1 Materials and Methods

Bacterial Strains

|0053] E. coli strains BL21 (DE3), E. coli BL21 (Rosetta), DB3.1 and DH5α are all commercially available from Novagen and Invitrogen, respectively. BL21 has the genotype: F^" ompT [loή\ hsdSβ (Γ_B ITI_B ; an E. coli B strain) with DE3, a λ prophage carrying the T7 RNA polymerase gene. DH5α has the genotype: F^", ΦS0dlacZM15, endAl, recAl, hsdRl 7 (rk^~, mk⁺), supE44, thi-\, gyrA96, relAl, A{lacZYA-argF)UJ69, λ^".

Destination Vectors

[0054] Plasmid pET43-DVbase is constructed by digesting pET43a (Novagen) with Ndel and Hindlll, and then inserting (via ligation) a DNA fragment created by annealing the two oligonucleotide primers SEQ ID NO:1 and SEQ ID NO:2.

SEQ ID NO:1 tatgagatct gatatcgcta gctagatagc taga

SEQ ID NO:2 agcttctagc tatctagcta gcgatatcag atctca

[0055] These clones are transformed into E. coli DH5α, selected on 100 μg/ml ampicillin, and verified by sequencing.

[0056] To construct pDest-590, pET43-DVbase is then digested with EcoRV, and the Gateway rfa cassette (Invitrogen) was ligated into the vector. The ligation mixture is then transformed into E. coli DB3.1 (Invitrogen) and selected on 100 μg/ml ampicillin and 15 μg/ml chloramphenicol.

[0057] Additional amino-terminal fusion destination vectors are constructed by digestion of pDest-590 with BgIIl, and ligation of BamHl digests of various PCR fragments for the different fusion proteins. The primer sequences used to amplify the fusion proteins for these vectors were: pDest-566 (Hisό-MBP)

[0058] This vector is capable of forming a fusion protein between a desired target gene, and gene sequences that encode the maltose binding protein of E. coli (Genbank Accession No. JOl 648; Roa, M. et al. (1980) "LOCATION OF A PHAGE BINDING REGION ON AN OUTER MEMBRANE PROTEIN," FEBS Lett. 121 (1): 127- 129; Bedouelle, H. et al ( 198O) ¹¹MUTATIONS WHICH ALTER THE FUNCTION OF THE SIGNAL SEQUENCE OF THE MALTOSE BINDING PROTEIN OF ESCHERICHIA COLL" Nature 285 (5760):78-81 ; Emr, S.D. et al (1980) "SEQUENCE ANALYSIS OF MUTATIONS THAT PREVENT EXPORT OF LAMBDA RECEPTOR, AN ESCHERICHIA COLI OUTER MEMBRANE PROTEIN," Nature 285 (5760):82-85; Hedgpeth, J. et al. (1980) "DNA SEQUENCE ENCODING THE NH2-TERMINAL PEPTIDE INVOLVED IN TRANSPORT OF LAMBDA RECEPTOR, AN ESCHERICHIA COLI SECRETORY PROTEIN," Proc. Natl. Acad. Sci. U.S.A. 77 (5):2621-2625; Clement, J.M. et al. (1981) "GENE SEQUENCE OF THE LAMBDA RECEPTOR, AN OUTER MEMBRANE PROTEIN OF E. COLi Kl 2," Cell 27 (3 Pt 2):507-514; Bedouelle, H. (1982) "STRUCTURE OF THE MALB REGULATORY INTERVAL," Ann. Inst. Pasteur Microbiol. 133: 65-70 (1982); Bedouelle, H. et al. (1982) "PROMOTERS OF THE MALEFG AND MALK-LAMB OPERONS IN ESCHERICHIA COLI Kl 2," J. MoI. Biol. 161 (4):519-531 ; Bedouelle, H. et al. () "A DNA SEQUENCE CONTAINING THE CONTROL REGIONS OF THE MALEFG AND MALK-LAMB OPERONS IN ESCHERICHIA COLI Kl 2," MoI. Gen. Genet. 185 (1), 82-87 (1982); Hall, M.N. et al (1982) "A ROLE FOR MRNA SECONDARY STRUCTURE IN THE CONTROL OF TRANSLATION INITIATION," Nature 295 (5850):616-618; Gilson, E. et al (1982) "SEQUENCE OF THE MALK GENE IN E.COLI K12," Nucleic Acids Res. 10 (22):7449-7458; Emr, S.D. et al (1983) "Importance of secondary structure in the signal sequence for protein secretion," Proc. Natl. Acad. Sci. U.S.A. 80 (15):4599-4603; Bankaitis, V.A. et al (1984) "INTRAGENIC SUPPRESSOR MUTATIONS THAT RESTORE EXPORT OF MALTOSE BINDING PROTEIN WITH A TRUNCATED SIGNAL PEPTIDE," Cell 37 (l):243-252; Duplay, P. et al (1984) "SEQUENCES OF THE MALE GENE AND OF ITS PRODUCT, THE MALTOSE- BINDING PROTEIN OF ESCHERICHIA COLI Kl 2," J. Biol. Chem. 259 (16): 10606-

10613; Froshauer, S. et al (1984) "THE NUCLEOTIDE SEQUENCE OF THE GENE FOR MALF PROTEIN, AN INNER MEMBRANE COMPONENT OF THE MALTOSE TRANSPORT SYSTEM OF ESCHERICHIA COLL REPEATED DNA SEQUENCES ARE FOUND IN THE MALE-MALF INTERCISTRONIC REGION, J. Biol. Chem. 259 (17), 10896-10903. The encoded fusion also contains six tandem histidine residues to facilitate purification.

|0059] pDest-566 (His6-MBP) is produced from pDest590 by ligating a BamHI digested PCR fragment containing the E. coli MBP gene. The MBP gene fragment was obtained from E. coli DNA via PCR using the primers:

Forward primer (SEQ ID NO:3) ccacccaccg gatcccatca ccatcaccat cacggcaaaa tcgaagaagg taaactgg

Reverse primer (SEQ ID NO:4) ccacccaccg gatcccgaat tagtctgcgc gtctttcagg gcttc

pDest-568 (DsbC fusion plasmid)

[0060| This vector is capable of forming a fusion protein between a desired target gene, and gene sequences that encode the E. coli DsbC gene (Genbank Accession No. U28375). The amino acid sequence of the DsbC protein is (SEQ ID NO:5): MKKGFMLFTL LAΆFSGFAQA DDAAIQQTLA KMGIKSSDIQ PAPVAGMKTV

LTNSGVLYIT DDGKHIIQGP MYDVSGTAPV NVTNKMLLKQ LNALEKEMIV

YKAPQEKHVI TVFTDITCGY CHKLHEQMAD YNALGITVRY LAFPRQGLDS

DAEKEMKAIW CAKDKNKAFD DVMAGKSVAP ASCDVDIADH YALGVQLGVS GTPAVVLSNG TLVPGYQPPK EMKEFLDEHQ KMTSGK

|0061] SEQ ID NO:5 contains the mature DsbC protein as well as the signal sequence responsible for mediating the transfer of the protein to the periplasm.

[0062] pDest-568 (DsbC) is produced from pDest590 by ligating a BamHI digested PCR fragment containing the DsbC gene (including its signal sequence). The E. coli DsbC fragment was obtained from E. coli DNA via PCR using the primers:

Forward primer (SEQ ID NO:6) agggactaag gatccaagaa aggttttatg ttgtttactt tg

Reverse primer (SEQ ID NO:7) cgagttagag gatcctttac cgctggtcat tttttggtgt teg [0063| These primers hybridize to the nucleotide sequence encoding the DsbC protein (SEQ ID NO:8) at sites shown in underline (for the Forward primer) or in double underline (for the Reverse primer), and therefore enable the cloning of the intervening region.

SEQ ID NO:8 atgaagaaag gttttatgtt gtttactttg ttagcggcgt tttcaggctt tgctcaggct gatgacgcgg caattcaaca aacgttagcc aaaatgggca tcaaaagcag cgatattcag cccgcgcctg tagctggcat gaagacagtt ctgactaaca gcggcgtgtt gtacatcacc gatgatggta aacatatcat ' tcaggggcca atgtatgacg ttagtggcac ggctccggtc aatgtcacca ataagatgct gttaaagcag ttgaatgcgc ttgaaaaaga gatgatcgtt tataaagcgc cgcaggaaaa acacgtcatc accgtgttta ctgatattac ctgtggttac tgccacaaac tgcatgagca aatggcagac tacaacgcgc tggggatcac cgtgcgttat cttgctttcc cgcgccaggg gctggacagc gatgcagaga aagaaatgaa agctatctgg tgtgcgaaag ataaaaacaa agcgtttgat gatgtgatgg caggtaaaag cgtcgcacca gccagttgcg acgtggatat tgccgaccat tacgcacttg gcgtccagct tggcgttagc ggtactccgg cagttgtgct gagcaatggc acacttgttc cgggttacca gccgccgaaa gagatgaaag aattcctcga cgaacaccn aaaatgacca αcσσtaaata a pDest-579 (signal sequence-less Skp)

[0064] This vector is capable of forming a fusion protein between a desired target gene, and gene sequences that encode the E. coli skp gene (Genbank Accession No. M21 1 18; Hoick, A. et al. (1988) "CLONING AND SEQUENCING OF THE GENE FOR THE DNA-BiNDiNG 17K PROTEIN OF ESCHERICHIA COLI," Gene 67 (1 ): 1 17- 124). The amino acid sequence of the Skp protein is (SEQ ID NO:9):

MKKWLLAAGL GLALATSAQA ADKIAIVNMG SLFQQVAQKT GVSNTLENEF

KGRASELQRM ETDLQAKMKK LQSMKAGSDR TKLEKDVMAQ RQTFAQKAQA

FEQDRARRSN EERGKLVTRI QTAVKSVANS QDI DLVVDAN AVAYNSSDVK DITADVLKQV K

[0065] The signal sequence (residues 1-17 of SEQ ID NO:9) is shown in bold and underlined.

[0066| pDest-579 (signal sequence-less Skp) is produced from pDest590 by ligating a BamHl digested PCR fragment containing the E. coli Skp gene (minus its signal sequence). The Skp fragment was obtained from E. coli DNA via PCR using the primers: Forward primer (SEQ ID NO:10) gcgagcgagg atccgctgac aaaattgcaa tcgtcaacat ggg

Reverse primer (SEQ ID NO: 11) aggctagcgg atcctttaac ctgtttcagt acgtcggcag

[0067] These primers hybridize to the nucleotide sequence encoding the Skp protein (SEQ ID NO: 12) at sites shown in underline (for the Forward primer) or in double underline (for the Reverse primer), and therefore enable the cloning of the intervening region: atgaaaaagt ggttattagc tgcaggtctc ggtttagcac tggcaacttc tgctcaggcg gctgacaaaa ttgcaatcgt caacatgggc agcctgttcc agcaggtagc gcagaaaacc ggtgtttcta acacgctgga aaatgagttc aaaggccgtg ccagcgaact gcagcgtatg gaaaccgatc tgcaggctaa aatgaaaaag ctgcagtcca tgaaagcggg cagcgatcgc actaagctgg aaaaagacgt gatggctcag cgccagactt ttgctcagaa agcgcaggct tttgagcagg atcgcgcacg tcgttccaac gaagaacgcg gcaaactggt tactcgtatc cagactgctg tgaaatccgt tgccaacagc caggatatcg atctggttgt tgatgcaaac gccgttgctt acaacagcag cgatgtaaaa αacatcactq ccσacσtact gaaacagqtt aaataa

[0068] After ligation, DNA is transformed into E. coli DB3.1 , selected on ampicillin and chloramphenicol, and plasmids are isolated from individual clones. These plasmids are then sequenced completely throughout the fusion protein to verify that the correct clone is generated in the correct orientation.

Cloning Procedure

[0069] Expression vectors with Skp, DsbC and MBP (maltose binding protein) proteins are made by introducing gene sequences encoding desired target proteins into the above-described destination vectors using standard recombination mediated cloning procedures (Invitrogen). For Skp, signal sequence is eliminated so that expression fusion proteins will be retained in the cytoplasm.

[0070] Several model genes that had been shown to be extremely difficult to express as soluble proteins are cloned in order to compare their expression and the effect of using different expression tags. In all cases, the same protocol is used to generate the clones using the Gateway system (Invitrogen). Genbank reference numbers for model target genes and primers used to amplify these model genes from human DNA are:

[0071] H. sapiens BΗD (Folliculin): AF517523 Forward primer (SEQ ID NO:13): ggggacaact ttgtacaaaa aagttggcac catgaatgcc atcgtggctc tctgccac

Reverse primer (SEQ ID NO:14): ggggacaact ttgtacaaga aagttggcta gttccgagac tccgaggctg tggggc

[0072] H. sapiens Wnt5a: NM_003392 Forward primer (SEQ ID NO: 15): aggtggctcg ggtgctggcc aggttgttat agaagctaat tc

Reverse primer (SEQ ID NO:16): ggggacaact ttgtacaaga aagttggcta tttgcacacg aactgatcca caatc

Adapter Primer (SEQ ID NO:17): ggggacaact ttgtacaaaa aagttggcga aaacctgtac ttccaaggtg gctcgggtgc tggc

[0073] H. sapiens Endostatin: AFl 84060 Forward primer (SEQ ID NO: 18): ggcgaaaacc tgtacttcca aggccacagc caccgcgact tccagccggt gc

Reverse primer (SEQ ID NO: 19): ggggacaact ttgtacaaga aagttggcta cttggaggca gtcatgaagc tg

Adapter Primer (SEQ ID NO:20): ggggacaact ttgtacaaaa aagttggcga aaacctgtac ttccaaggc [0074] Y. pestis YopD: CAB54905

Forward primer (SEQ ID NO:21): ggcgaaaacc tgtacttcca aacaataaat atcaagacag acagccc

Reverse primer (SEQ ID NO:22): ggggacaact ttgtacaaga aagttggcta gacaacacca aaagcggctt tcatgg

Adapter Primer (SEQ ID NO:23): ggggacaact ttgtacaaaa aagttggcga aaacctgtac ttccaaggc

[0075] H. sapiens Hiβa: NM OO J 530

Forward primer (SEQ ID NO:24): ggcgaaaacc tgtacttcca aggcatggag ggcgccggcg gcgcgaacga c

Reverse primer (SEQ ID NO:25): ggggacaact ttgtacaaga aagttggcta gttaacttga tccaaagctc tgag

Adapter Primer (SEQ ID NO:26): ggggacaact ttgtacaaaa aagttggcga aaacctgtac ttccaaggc

[0076] H. sapiens IL-13: NM_002188

Forward primer (SEQ ID NO:27): ggcgaaaacc tgtacttcca aggcggtccg gttccgccgt ctaccgcgc

Reverse primer (SEQ ID NO:28): ggggacaact ttgtacaaga aagttggcta cgcgttgaaa cgaccttcac gg

Adapter Primer (SEQ ID NO:29): ggggacaact ttgtacaaaa aagttggcga aaacctgtac ttccaaggc |0077| H. sapiens IFNa -hyb3: (Schmeisser, H. et al. "AMINO ACID SUBSTITUTIONS IN LOOP BC AND HELIX C AFFECT ANTIGENIC PROPERTIES OF HELIX

D IN HYBRID 1FN-ALPHA21 A/ALPHA2C MOLECULES," J Interferon Cytokine Res. 22(4):463-472.

Forward primer (SEQ ID NO:30): ggcgaaaacc tgtacttcca aggctgtgat ctgcctcaaa cccacagcc

Reverse primer (SEQ ID NO:31): ggggacaact ttgtacaaga aagttggtta ttccttcctc cttaatcttt cttg

Adapter primer (SEQ ID NO:32): ggggacaact ttgtacaaaa aagttggcga aaacctgtac ttccaaggc

[0078] The destination vectors, pDest 566 (maltose binding protein-fusion vector), pDest 579 (Skp-Fusion vector) and pDest 568 (DsbC fusion vector), described above are used to clone the model genes. In brief, genes are amplified from cDNA or genomic DNA using primers containing Gateway recombination signal sequences. In many cases, a TEV protease cleavage site is introduced in front of the gene of interest to allow cleavage of the fusion protein after expression. In these cases, a second "adapter" primer is employed during amplification. The start and stop primers are added in the initial PCR reaction (200 nM each in a 50 μl reaction), along with 100-200 ng of template DNA. After 5 cycles of amplification, 200 nM adapter primers are added to the reactions, and amplification is continued for an additional 15 cycles. In cases where no adapter primer is needed, PCR is carried out for 15 cycles total. PCR cycling conditions are: 95°C, 30 sec; 55°C, 30 sec, 72⁰C, 1 min per Kb product. After amplification, the PCR products are cleaned using Qiagen's PCR Purification columns, and DNA is introduced into a Gateway BP reaction. 1 μl of PCR product is mixed with 150 ng of pDonr223 vector (Invitrogen) in a 20 μl reaction, and reacted with 4 μl BP Clonase for 1 hour at 30 °C. After stopping the reaction with Proteinase K, 1 μl is transformed into E. coli DH5a and samples were plated on LB with 50 μg/ml spectinomycin to select for correct clones. Several clones are grown, and plasmid DNA is prepared by alkaline lysis or FastPlasmid (Brinkmann). Clones are then sequence verified to ensure that no mutations had been introduced.

[0079] Using the above-described primers, genes encoding Folliculin, Wnt5a, IFN- Hyb3, Endostatin, YopD, Hiflα, IL- 13 and a Folliculin domain are cloned into the Skp fusion destination vector, genes encoding Folliculin, Wnt5a, IFN-Hyb3, Endostatin, YopD and a Folliculin domain are cloned into the MBP fusion destination vector and genes encoding Folliculin, Wnt5a and IFN-Hyb3 are cloned into the DsbC fusion destination vector. Cloning is accomplished according to the protocol of Gateway LR reaction (Invitrogen). A portion of the reaction (1 μl) is used to transform E. coli DH5α and plated onto ampicillin containing plates. Individual clones are digested with BsrGI to check for correct insert size. Correct clones were saved.

Growth And Expression Of Fusion Constructs

[0080] For growth and expression, 100 μl competent BL21 DE3 (Rosetta) was transformed with l μl each plasmid (fusion construct). After 20 min. on ice, the transformation mixture was heat shocked at 42°C for 45 seconds. Finally, 900 μl SOC is added and incubated at 30°C for 1 hr. To check the efficiency of transformation, 100 μl of the culture are plated onto ampicillin (100 μg/ml) and chloramphenicol (15 μg/ml) plates and the plates are incubated at 37°C. To the rest, 1 ml of SOC and 100 μg/ml ampicillin and 15 μg/ml chloramphenicol are added. The culture is grown overnight at 30°C .

Expression [0081] A single colony of each clone is grown in Circle Grow growth medium (Q- Biogene, California)) with amp (100 ug/ml) and chloramphenicol (15 μg/ml) at 30⁰C for 15 hrs. For expression, 450 μl of an overnight grown culture is inoculated into 15 ml Circle Grow (Q-Biogene) media containing ampicillin (100 μg/ml) and chloramphenicol (15 μg/ml) and grown at 30°C for approximately 4 hrs to an As₆₀ of 0.7-0.8. The culture is induced with 1 mM isopropyl-β-D- thiogalactopyranoside (IPTG) for 3 hr. at 3O⁰C and then harvested by centrifugation. The cultures, if not used immediately, are stored at -80°C.

[0082] Cells from the centrifuged 15 ml culture are suspended in 1 ml of Buffer (50 mM Tris, pH8, 50 mM NaCl). 100 μl of protease inhibitor (Roche, 1 tablet dissolved in 600 μl of the Buffer) is added to the cell suspension before sonication with a micro-tip (3 times for 10 sec each at 65% efficiency using a micro-tip probe). A portion (50-100 μl) is saved to permit a determination of total (T) protein expression. The remaining material is centrifuged for 5 min at maximum speed (14,000-15,000 rpm) in a cold microcentrifuge (Eppendorf). The supernatant is saved and analyzed for soluble (S) expression of the test protein. Usually, 1-1.5 μl of the sample is used for polyacrylamide gel analysis (4-20%, Invitrogen). The gel is stained with Coomassie blue.

Polyacrylamide gel analysis

[0083J For typical analysis, 1-2 μl of the extract, total or soluble, is loaded onto a 4-20% polyacrylamide gel (Invitrogen). The gel is stained with Coomassie stain (Fast-stain, Invitrogen), de-stained and photographed.

Example 2 Production of Recombinant Protein as Skp Fusions |0084] Using the materials and methods described above, Skp fusion vectors are prepared and used to express Wnt5a, Folliculin, IFN-Hyb3, Endostatin and YopD as Skp fusions.

Skp fusion of Wnt5a, Folliculin and IFN-Hyb3

|0085] BL21 DE3 (Rosetta) containing the plasmid is grown as described in the Example 1. Figure 1 shows the results of efforts to express Wnt5a, Folliculin and IFN-Hyb3 as Skp fusions. As shown in Figure 1, Wnt5a with Skp fusion produces almost 40% of the total protein of which approximately 15% is soluble. Similarly, Folliculin is expressed extremely well as almost 30% of the total protein. The soluble fraction of Skp-folliculin is estimated to be about 40% of total Folliculin protein. For IFN-H yn3 fusion, total amount of expressed protein was about 25% of which about 25% was soluble.

Skp fusion of YopD and Endostatin

[0086] Figure 2 shows the expression of YopD and Endostatin as Skp fusion. YopD is expressed as 5-10% of total protein of which about 30-40% is soluble. Skp-Endostatin fusion produces almost 50% of total protein. However, about 15% is soluble.

Skp fusion of Hiflα, IL-13 and Folliculin domain

[0087] Figure 3 shows the expression of total (T) and soluble (S) proteins Hifl A, IL-13, a domain of Folliculin (FD) and full-length Folliculin (F) using a Skp fusion expression system. As shown in Figure 3, signal sequence-less Skp fusion of IL- 13 and a domain of Folliculin produces 40 and 50% of total protein, respectively. However, about 30% of ILl 3 and 25% of Folliculin domains are soluble. A repeat experiment with Folliculin produces the same result as shown in Figure 1. The only protein that is produced in almost 100% insoluble form was Hiflα, although the level of expression is quite good.

Example 3 Production of Recombinant Protein as MBP Fusions [0088] Using the materials and methods described above, MBP fusion vectors are prepared and used to express, Folliculin, IFN-Hyb3, Wnt5a YopD and Endostatin as MBP fusions. Figure 4 shows the results of efforts to express Folliculin, IFN- Hyb3, Wnt5a, YopD and Endostatin as MBP-fusion. As can be seen from Figure 4, the level of expression of Folliculin, IFN-Hyb3, Wnt5a, YopD and Endostatin are 15%, 20%, 20%, 20% and 40%, respectively. Of the total amount of fusion protein, the amount of soluble fusion protein for Folliculin, IFN-Hyb3, Wnt5a, YopD and Endostatin is estimated to be about 40%, 25%, 5%, 50% and 30%, respectively. Examplc 4 Production of Recombinant Protein as DsbC Fusions

|0089] Using the materials and methods described above, DsbC fusion vectors are prepared and used to express, Folliculin, 1FN-Hyb3, and Wnt5a as DsbC fusions. In this case, the fusion protein will be accumulated in the periplasmic space.

Figure 5 shows the results of efforts to express Folliculin, IFN-Hyb3 and Wnt5a as DsbC fusions. For DsbC fusion, the level of expression for all target proteins is lower compared to Skp or MBP fusion.

Example 5 Solubility of Recombinant Proteins

[0090] As stated above, the model genes chosen are extremely difficult to express as soluble proteins. The above examples demonstrate that success is seen when these genes are expressed as fusion protein with MBP. It has been suggested that maltose binding protein (MBP), as fusion partner is a potent solubility enhancer (Fox et al. (2003) "MALTODEXTRIN-BINDING PROTEINS FROM DIVERSE BACTERIA AND ARCHAEA ARE POTENT SOLUBILITY ENHANCERS," FEBS Letters 537:53-57). Therefore, the soluble expression of some of the model proteins was compared with MBP.

[0091] The levels of expression for the DsbC fusions are compared to those obtained from the Skp and MBP fusions. Overall protein synthesis (total or "T") as well as soluble ("S") protein synthesis for DsbC fusions was generally less compared to either Skp or MBP. This is perhaps due to the final destination of the fusion protein, which in this case is periplasm. The only exception was IFN-Hyb3 fusion protein where almost 90% of the fusion protein was expressed as soluble protein. Comparative data obtained by scanning the gels of Figures 1-5 are summarized in Table 1.

[0092] The data show that the expression systems of the present invention are capable of expressing high levels of soluble protein.

[0093] Signal sequence-less Skp fusions are found to produce more overall protein (ranging from 25-40% of the total protein), except for YopD. In addition, Skp fusions are found to produce more soluble protein compared to MBP and DsbC fusion. For Endostatin fusions, MBP seem to produce more soluble protein.

[0094] One factor that should be considered when estimating the amount of protein produced as a fusion protein is the contribution of the fusion partner in terms of molecular weight. MBP is about 43 Kd where as the size of Skp is 17 Kd. For example, if the size of the test protein is 20 Kd and if both fusion produced 10 μg protein, the actual amount of the protein of interest (20 Kd) would be about 5.5 μg for Skp-fusion and 3 μg for MBP-fusion, which is almost half compared to Skp fusion. Figure 6 illustrates the comparative production of total (T) and soluble (S) expression of proteins using an aminoterminal Skp or DsbC fusion.

Example 6 T7 Protein Kinase Fusions |0095] For certain proteins, problems of insolubility are so severe that even the use of chaperone fusion proteins Skp/DsbC still results in a significant accumulation of insoluble material. Folliculin, for example, is highly insoluble when expressed in E. coli. As demonstrated in Figure 7, even in the presence of various chaperones, GroEL- ES/Trigger factor (TF) or Skp/DsbC, a significant proportion of Folliculin remained insoluble.

[0096J To address such situations, and further increase the yield of soluble protein, the T7 protein kinase is used in accordance with the principles of the present invention to facilitate the expression and solubility of fusion partners. Since the carboxy terminal domain of the T7 protein kinase is toxic to E. coli (participating in the shut-off of host transcription), it is preferred to employ (as a fusion partner of the target protein) a fragment of the T7 protein kinase that lacks the toxic portion of the complete protein. Alternatively, the complete T7 protein may be used as a fusion molecule, preferably with one or more residues thereof mutated to eliminate or reduce the toxicity.

[0097] It is particularly preferred to employ the amino terminal domain of the T7 protein kinase to facilitate the expression and solubility of fusion partners. SEQ ID NO:33 is a polynucleotide encoding the complete phage T7 protein kinase (SEQ ID NO:34).

SEQ ID NO:33: atgaacatta ccgacatcat gaacgctatc gacgcaatca aagcactgcc aatctgtgaa cttgacaagc gtcaaggtat gcttatcgac ttactggtcg agatggtcaa cagcgagacg tgtgatggcg agctaaccga actaaatcag gcacttgagc atcaagattg gtggactacc ttgaagtgtc tcacggctga cgcagggttc aagatgctcg gtaatggtca cttctcggct gcttatagtc acccgctgct acctaacaga gtgattaagg tgggctttaa gaaagaggat tcaggcgcag cctataccgc attctgccgc atgtatcagg gtcgtcctgg tatccctaac gtctacgatg tacagcgcca cgctggatgc tatacggtgg tacttgacgc acttaaggat tgcgagcgtt tcaacaatga tgcccattat aaatacgctg agattgcaag cgacatcatt gattgcaatt cggatgagca tgatgagtta actggatggg atggtgagtt tgttgaaact tgtaaactaa tccgcaagtt ctttgagggc atcgcctcat tcgacatgca tagcgggaac atcatgttct caaatggaga cgtaccatac atcaccgacc cggtatcatt ctcgcagaag aaagacggtg gcgcattcag catcgaccct gaggaactca tcaaggaagt cgaggaagtc gcacgacaga aagaaattga ccgcgctaag gcccgtaaag aacgtcacga ggggcgctta gaggcacgca gattcaaacg tcgcaaccgc aaggcacgta aagcacacaa agctaagcgc gaaagaatgc ttgctgcgtg gcgatgggct gaacgtcaag aacggcgtaa ccatgaggta gctgtagatg tactaggaag aaccaataac gctatgctct gggtcaacat gttctctggg gactttaagg cgcttgagga acgaatcgcg ctgcactggc gtaatgctga ccggatggct atcgctaatg gtcttacgct caacattgat aagcaacttg acgcaatgtt aatgggctga SEQ ID NO:34:

MNITDIMNAI DAIKALPICE LDKRQGMLID LLVEMVNSET CDGELTELNQ ALEHQDWWTT LKCLTADAGF KMLGNGHFSA AYSHPLLPNR VIKVGFKKED SGAAYTAFCR MYQGRPGIPN VYDVQRHAGC YTVVLDALKD CERFNNDAHY KYAEIASDII DCNSDEHDEL TGWDGEFVET CKLIRKFFEG IASFDMHSGN IMFSNGDVPY ITDPVSFSQK KDGGAFSIDP EELIKEVEEV ARQKEIDRAK ARKERHEGRL EARRFKRRNR KARKAHKAKR ERMLAAWRWA ERQERRNHEV AVDVLGRTNN AMLWVNMFSG DFKALEERIA LHWRNADRMA IANGLTLNID KQLDAMLMG [0098] SEQ ID NO:35 is the preferred polynucleotide encoding a preferred amino terminal phage T7 protein kinase domain (SEQ ID NO:36) used in the PK fusions of the present application (in which the first and second residues of the phage T7 protein kinase are separated by the dipeptide RS).

SEQ ID NO:35: atgagatcca acattaccga catcatgaac gctatcgacg caatcaaagc actgccaatc tgtgaacttg acaagcgtca aggtatgctt atcgacttac tggtcgagat ggtcaacagc gagacgtgtg atggcgagct aaccgaacta aatcaggcac ttgagcatca agattggtgg actaccttga agtgtctcac ggctgacgca gggttcaaga tgctcggtaa tggtcacttc tcggctgctt atagtcaccc gctgctacct aacagagtga ttaaggtggg ctttaagaaa gaggattcag gcgcagccta taccgcattc tgccgcatgt atcagggtcg tcctggtatc cctaacgtct acgatgtaca gcgccacgct ggatgctata cggtggtact tgacgcactt aaggattgcg agcgtttcaa caatgatgcc cattataaat acgctgagat tgcaagcgac atcattgatt gcaattcgga tgagcatgat gagttaactg gatgggatgg tgagtttgtt gaaacttgta aactaatccg caagttcttt gagggcatcg cctcattcga catgcatagc gggaacatca tgttctcaaa tggagacgta ccatacatca ccgacccggt atcattctcg cagaagaaag acggtggcgc attcagcatc gaccctgagg aactcatcaa ggaagtcgag gaagtcgcac ga

SEQ ID NO:36:

MRSNiTDiMNAi DAIKΆLPICE LDKRQGMLID LLVEMVNSET

CDGELTELNQ ALEHQDWWTT LKCLTADAGF KMLGNGHFSA

AYSHPLLPNR VIKVGFKKED SGAAYTAFCR MYQGRPGI PN

VYDVQRHAGC YTVVLDALKD CERFNNDAHY KYAEIAS DI I DCNSDEHDEL TGWDGEFVET CKLIRKFFEG IAS FDMHSGN IMFSNGDVPY ITDPVSFSQK KDGGAFSIDP EELIKEVEEV AR

[0099J Bacteriophage T7 (T-PK) early gene 0.7 encodes two activities. About two-third of amino end of the protein encodes protein kinase activity, while the C- terminal one-third end is responsible for host transcription shutoff following infection (Brunoskis, 1. et al. (1977) "THE PROCESS OF INFECTION WITH BACTERIOPHAGE T7. VI. A PHAGE GENE CONTROLLING SHUTOFF OF HOST RNA SYNTHESIS," Virology 50:322-327; Rothman-Denes, L.B. et al. (1973) "A T7 GENE FUNCTION REQUIRED FOR SHUT-OFF OF HOST AND EARLY T7 TRANSCRIPTION," In Fox, CF. , Robinson, W. S (Eds), Virus Research; Academic Press, New York, pp. 227-239; McAllister, W.T. et al. (1977) "ROLES OF EARLY GENES OF BACTERIOPHAGE T7 IN SHUTOFF OF HOST MACROMOLECULAR SYNTHESIS," J. Virology 23:543-553). Although full-length T-PK protein is toxic and cannot be expressed in E. coli, a polypeptide lacking the C-terminal end can be cloned in a plasmid and overexpressed at a very high level in a completely soluble form (Michalewicz, J. et al. (1992) "MOLECULAR CLONING AND EXPRESSION OF BACTERIOPHAGE T7 0.7 (PROTEIN KINASE) GENE," Virology 186:452-562; Warren, M.B. et al. (2004) "EXPRESSION OF BiRT-HOGG-DUBE GENE MRNA IN NORMAL AND NEOPLASTIC HUMAN TISSUES," Mod Pathol. 17(8):998-101 1). In addition, it has been shown that when cloned as a single copy gene in E. coli chromosome, the gene is expressed at almost 40% of total E. coli protein (Warren, M.B. et al. (2004) "EXPRESSION OF BIRT-HOGG-DUBE GENE MRNA IN NORMAL AND NEOPLASTIC HUMAN TISSUES," Mod Pathol. 17(8):998-101 1). This high level of protein synthesis is dependent on protein kinase activity and it stimulates its own synthesis (auto-enhancement) both at the transcription and post-transcriptional level (Warren, M. B. et al. (2004) "EXPRESSION OF BiRT-HoGG-DUBE GENE MRNA IN

NORMAL AND NEOPLASTIC HUMAN TISSUES," Mod Pathol. 17(8):998-l 01 1 ). In addition, RNaseE, the major endonuclease to control mRNA, has been shown to be heavily phosphorylated by T-PK (Warren, M.B. et al. (2004) "EXPRESSION OF BlRT-HOGG-DUBE GENE MRNA IN NORMAL AND NEOPLASTIC HUMAN TISSUES," Mod Pathol. 17(8):998-101 1) and may impair RNaseE activity and stabilize mRNA. These intriguing properties of T-PK compelled us to test whether it could be a fusion partner for high expression and soluble protein synthesis.

[00100] The fusion is prepared by fusing a polynucleotide sequence encoding the PK to a polynucleotide encoding the target protein. Preferably, the PK-encoding sequence will be fused to the amino terminus of the polynucleotide encoding the target protein. In a preferred embodiment, a protease recognition site will be included between the PK and target protein encoding sequences so as to permit the cleavage of the chaperone protein from the target protein. A preferred protease recognition site is the site recognized by the 27 kDa catalytic domain of the Nuclear Inclusion a (NIa) protein of the tobacco etch virus ("TEV protease"). Sequences of suitable TEV protease recognition sites are disclosed by Carrington, J. C. et al. (1988) ["A VIRAL CLEAVAGE SITE CASSETTE: IDENTIFICATION OF AMINO ACID SEQUENCES REQUIRED FOR TOBACCO ETCH VIRUS POLYPROTEIN PROCESSING" Proc. Natl. Acad. Sci. USA 85:3391 -3395]; Dougherty, W. et al. (1989) ["Molecular Genetic Analysis Of A Plant Virus Polyprotein Cleavage Site: A Model," Virology 171 :356-364]; Parks, T. et al. (1992) ["Cleavage profiles of tobacco etch virus (TEV)-derived substrates mediated by precursor and processed forms of the TEV NIa proteinase," J. Gen. Virol. 73:149-155]; and by Dougherty, W. et al. ( 1993) ["Expression of Virus-Encoded Proteinases: Functional and Structural Similarities with Cellular Enzymes," Microbiol. Rev. 57: 781 -822]. A preferred TEV protease recognition sites has the sequence (the protease cleaves between the Q and G residues):

SEQ ID NO:37 ELNYFQG 100101] '^{n a} further embodiment, a polynucleotide encoding an affinity tag is also included so as to peπnit the facile recovery of the expressed fusion protein. Figure 8 illustrates the configuration of preferred PK fusion proteins.

[00102J Fusion of PK enhances the level of production of passenger protein substantially even when compared maltose binding protein (MBP), one of the most popular fusion partner. PK also enhances solubility some of the most insoluble proteins. In addition, the fusion also enhances the synthesis of protein where no synthesis was detected with MBP. Figure 9 shows the vector diagram of a preferred PK vector (pDest590-d-PK), and the ability of the vector to facilitate the soluble expression of Hifla, Folliculin, a Folliculin domain (FD) and ILl 3. All of these proteins are highly insoluble when expressed in E. coli. However, T7 protein kinase fusion of these proteins improved solubility quite remarkably, except for Hifla, where approximately 10% solubility was obtained. However, almost 100% solubility of the Folliculin domain, 90% solubility of Folliculin and 25% solubility of ILl 3 were obtained.

[00103] To facilitate the purification of the T7 protein kinase (which cannot generally be purified except through conventional procedures), an affinity tag ("Strep-tag II") (Sigma Genosys) was incorporated into the fusion. As shown in Figure 9, the level of expression and solubility are similar with or without the affinity tag. One-step purification of the fusion protein was done using a Strep- tactin column matrix (IDA, Germany). A portion of soluble fraction (L) of the fusion protein was loaded onto the column (200 μl). The flow-through fraction (F) was collected. The column was washed with a five-column volume of wash buffer (supplied the vendor). Finally, the fusion protein was eluted with 12 column volume six times (elution tubes 1-6). All fractions were tested by polyacryl amide gel electrophoresis (Figure 10). A major full-length fusion protein eluted in fraction 2 and 3. Along with full-length Folliculin, some proteolytic cleavage products may have been purified. Figure 11 shows the corresponding purification of a fusion of PK and the DEFG Folliculin Fragment using a Strep-tactin column matrix (IDA, Germany). [00104] To find out if the fusion protein can be separated from the fusion partner, a portion of purified fusion protein (fraction 3) was digested with two different amount (0.5% and 1 % v/v) of TEV protease (2.5 μg/μl). Digestion was carried out at 30°C for 1 hr and analyzed by 4-20% polyacrylamide gel electrophoresis. As shown in Figure 12, almost all of Folliculin domain (DEFG) can be separated by TEV protease. However, only a small fraction of full-length Folliculin fusion protein can be digested by TEV. Note that in this case, there was no spacer between the TEV protease site and the fusion protein. It is possible that full-length folded Folliculin somehow masked the TEV protease site and that complete cleavage therefore did not occur. It is preferred to form Folliculin fusion proteins with 5 and 10 amino acids spacer regions between the TEV protease site and the fusion protein.

[00105] Figure 13 shows a comparison of the level of expression and solubility of Folliculin with Strepll-Skp or StrepII-PK fusion partners (Tev5 and TevlO Fusions). Strep-tagll (IBA Incorporated, Germany) is the affinity purification tag and PK is the T7 kinase fusion tag. The sequence of Strep-tagll is:

SEQ ID NO:38 NWSHPQFEK

[00106] In order to purify the fusion protein, a purification tag has been incorporated. However, purification can be accomplished using affinity tags other than Strep-tag. For example, His-tag, Flag-tag, calmodulin-tag, etc. can be used in lieu of Strep-tag. Tev-5 and Tev-10 describe the linker between the TEV protease site and the beginning of the protein. The sequences of Tev-5 and Tev-10 are provided below (SEQ ID NO: 39 and SEQ ID NO: 40, respectively; the underlined residues are the TEV protease recognition site)

SEQ ID NO: 39 ENLYFQGGSG AG

SEQ ID NO: 40 ENLYFQGSGA GGSGAG

[00107] The inclusion of the TEV protease recognition site will result in the release of the fusion partner from the proteins of interest by TEV protease. Skp was shown to be a better fusion partner than MBP (maltose binding protein) for both expression as well as solubility. The results shown in Figure 13 indicate that T7 Protein Kinase fusion produced more total protein as well as soluble compared to Skp fusion. Thus, T7 Protein Kinase, appears to be the best "soluble expression tag" of the present invention. Figure 14 shows the corresponding purification of Strep-tag Il-PK-Folliculin TevlO fusion protein. Purification was done as described above.

[00108] TEV protease cleavage was done as described above (see Figure 12). Interestingly, with spacer region installed between the TEV protease cleavage site and the fusion protein more efficient cleavage occurred. However, optimal amounts of TEV protease and reaction condition are desired in order to obtain complete removal of the fusion partner. Figure 15 shows TEV protease digestion of a StreplI-PK-TEV10- Folliculin fusion molecule.

[00109] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[00110] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Claims

What is Claimed Is:

Claim 1. A nucleic acid molecule encoding a fusion protein, wherein said fusion protein comprises a solubility facilitating protein, or a fragment thereof, linked to a desired target protein.

Claim 2. The nucleic acid molecule of claim 1 , wherein said fusion protein encodes a fragment of said solubility facilitating protein.

Claim 3. The nucleic acid molecule of claim 1 , wherein said solubility facilitating protein is phage T7 protein kinase.

Claim 4. The nucleic acid molecule of claim 1 , wherein said solubility facilitating protein is a fragment of a phage T7 protein kinase.

Claim 5. The nucleic acid molecule of claim 4, wherein said fragment of said phage T7 protein kinase is an amino terminal fragment.

Claim 6. The nucleic acid molecule of claim 1 , wherein said solubility facilitating protein is a chaperone protein.

Claim 7. The nucleic acid molecule of claim 6, wherein said chaperone protein, or said fragment thereof lacks a chaperone protein signal sequence.

Claim 8. The nucleic acid molecule of claim 6, wherein said chaperone protein is selected from the group consisting of E. coli DsbC protein and E. coli Skp protein.

Claim 9. The nucleic acid molecule of claim 1 , wherein said fusion protein additionally comprises an affinity tag.

Claim 10. The nucleic acid molecule of claim 1 , wherein said nucleic acid molecule is a plasmid vector capable of replicating in a host cell.

Claim 1 1. The nucleic acid molecule of claim 10, wherein said host cell is an E. coli host cell.

Claim 12. A method of producing a desired target protein, wherein said method comprises the steps:

(A) introducing a plasmid vector capable of being expressed in a host cell into said host cell, wherein said vector comprises a nucleic acid molecule encoding a fusion protein, wherein said fusion protein comprises a solubility facilitating protein, or a fragment thereof, linked to said desired target protein;

(B) permitting said host cell to express said fusion protein;

(C) recovering said fusion protein from the cytoplasm of said host cell; and

(D) recovering said desired target protein from said fusion protein.

Claim 13. The method of claim 12, wherein said fusion protein encodes a fragment of said solubility facilitatingprotein.

Claim 14. The method of claim 12, wherein said solubility facilitating protein is phage T7 protein kinase.

Claim 15. The method of claim 12, wherein said solubility facilitating protein is a fragment of a phage T7 protein kinase.

Claim 16. The method of claim 15, wherein said fragment of said phage T7 protein kinase is an amino terminal fragment.

Claim 17. The method of claim 12, wherein said solubility facilitating protein is a chaperone protein.

Claim 18. The method of claim 17, wherein said chaperone protein, or said fragment thereof lacks a chaperone protein signal sequence.

Claim 19. The method of claim 17, wherein said chaperone protein is selected from the group consisting of E. coli DsbC protein and E. coli Skp protein.

Claim 20. The method of claim 12, wherein said fusion protein additionally comprises an affinity tag.

Claim 21. The method of claim 12, wherein said nucleic acid molecule is a plasmid vector capable of replicating in a host cell.

Claim 22. The method of claim 21 , wherein said host cell is an E. coli host cell.

Claim 23. A desired target protein produced by the process of:

(A) introducing a plasmid vector capable of being expressed in a host cell into said host cell, wherein said vector comprises a nucleic acid molecule encoding a fusion protein, wherein said fusion protein comprises a solubility facilitating protein, or a fragment thereof, linked to said desired target protein;

(B) permitting said host cell to express said fusion protein;

(C) recovering said fusion protein from the cytoplasm of said host cell; and

(D) obtaining said desired target protein from said fusion protein.

Claim 24. The desired target protein of claim 23, wherein said fusion protein encodes a fragment of said solubility facilitating protein.

Claim 25. The desired target protein of claim 23, wherein said solubility facilitating protein is phage T7 protein kinase.

Claim 26. The desired target protein of claim 23, wherein said solubility facilitating protein is a fragment of a phage T7 protein kinase.

Claim 27. The desired target protein of claim 26, wherein said fragment of said phage T7 protein kinase is an amino terminal fragment.

Claim 28. The desired target protein of claim 23, wherein said solubility facilitating protein is a chaperone protein.

Claim 29. The desired target protein of claim 28, wherein said chaperone protein, or said fragment thereof lacks a chaperone protein signal sequence.

Claim 30. The desired target protein of claim 28, wherein said chaperone protein is selected from the group consisting of E. coli DsbC protein and E. coli Skp protein.

Claim 31. The desired target protein of claim 23, wherein said fusion protein additionally comprises an affinity tag.

Claim 32. The desired target protein of claim 23, wherein said nucleic acid molecule is a plasmid vector capable of replicating in a host cell.

Claim 33. The desired target protein of claim 32, wherein said host cell is an E. coli host cell.