Process for production of recombinant proteins as a soluble form

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A target protein is prepared as soluble protein using a recombinant protein expression system. An expression vector is used that includes (1) an expression-inducible promoter sequence; (2) a first coding sequence including a polynucleotide coding for a polypeptide that is represented by the formula (Z)n; and (3) a second coding sequence that includes a polynucleotide that codes for a target protein. A method of producing the target protein is also used that includes expressing protein using this expression vector.

1. Field of the Invention

The invention relates to a method of producing a target protein as soluble protein.

2. Related Art

A large number of recombinant protein expression systems have been developed to date, including, for example, cell-free translation systems and recombinant protein expression systems within hosts such as bacteria, yeast, insects, transgenic animals, and transgenic plants. Escherichia coli is widely used as an expression system for heterologous protein because it is easily grown to high densities and because of the progress in research on host vector systems.

However, when a target protein is expressed using these recombinant protein expression systems, incorrect folding by the expressed protein can prevent expression of the functionality of the original protein and can result in the not insignificant formation of insoluble aggregates, known as inclusion bodies. Even when, for example, refolding is carried out in such cases after solubilization of the inclusion body with a denaturant or surfactant, the correctly folded protein exhibiting its native functionality is not necessarily obtained. In addition, even when protein expressing its original functionality is obtained, in many instances a satisfactory recovery rate is not obtained.

Against this background, a method of suppressing the formation of inclusion bodies of an expressed recombinant target protein has not been established to date. As a stand in for such a method, expression as a soluble protein is attempted by fusing the insoluble target protein with the soluble high molecular weight (40,000) maltose-binding protein or glutathione S-transferase (GST) (Fox, J. D. and Waugh, D. S., “Maltose-binding protein as a solubility enhancer.” METHODS MOL. BIOL., 205: 99-117 (2003); Ausubel, F. M. et al., editors, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol. 2, 16.0.1 (1996)). However, there have been problems such as, for example, the soluble protein may not exhibit its original activity or functionality and the target protein may become insoluble when the maltose-binding protein or GST is removed.

The ZZ domain is a synthetic IgG binding region developed on the basis of the IgG binding region of protein A (refer, for example, to Nilsson B. et al., Protein Eng., 1: 107-113 (1987)).

However, in those cases where the ZZ domain of the IgG binding region has been expressed fused with a target protein, there have been no reports of an effect whereby the solubility of the fusion protein is increased, nor have there been reports of an activity that contributes to an efficient refolding of the target protein to the active form of the protein. Up to the present time, the use of the ZZ domain originating from protein A has not gone beyond use, after expression of the fusion protein with a target protein, as a ligand in IgG antibody affinity chromatography in target protein purification. In addition, IgG antibody columns are expensive and there are only limited instances where they can be used even when a genetic recombinant fusion protein utilizing the ZZ domain is employed in mass production.

The Target Protein

There are no particular limitations on the target protein in the fusion protein of the invention. For example, even proteins that are prone to form inclusion bodies when expressed in a recombinant protein expression system can be advantageously used.

The target protein in the invention can be exemplified by protein (viral antigen), e.g., coat protein, core protein, protease, reverse transcriptase, integrase, and so forth, encoded in the genome of a pathogenic virus, e.g., hepatitis B virus, hepatitis C virus, HIV, influenza, and so forth; the Fab and (Fab)2 of antibodies; growth factors such as platelet-derived growth factor (PDGF), stem cell growth factor (SCF), hepatocyte growth factor (HGF), transforming growth factor (TGF), nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), and so forth; cytokines such as tumor necrosis factor, interferon, interleukin, and so forth; hematopoietic factors such as erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, thrombopoietin, and so forth; peptide hormones such as luteinizing hormone-releasing hormone (LH-RH), thyrotropin-releasing hormone (TRH), insulin, somatostatin, growth hormone, prolactin, adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), vasopressin, oxytoxin, calcitonin, parathyroid hormone (PTH), glucagon, gastrin, secretin, pancreozymin, cholecystokinin, angiotensin, human placenta lactogen, human chorionic gonadotropin (HCG), cerulein, motilin, and so forth; analgesic peptides such as enkephalin, endorphin, dynorphin, kyotorphin, and so forth; enzymes such as superoxide dismutase (SOD), urokinase, tissue plasminogen activator (TPA), asparaginase, kallikrein, and so forth; peptide neurotransmitters such as bombesin, neutrotensin, bradykinin, substance P, and so forth; as well as albumin, collagen, proinsulin, renin, al antitrypsin, and so forth. However, the target protein is not limited to the foregoing.