Gene expression technique

Abstract

The present invention provides a method for producing a desired protein (such as a desired heterologous protein) comprising: (a) providing a host cell comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein, a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein and a third gene, such as a third recombinant gene, encoding a desired protein (such as a desired heterologous protein), wherein the first and second chaperones are different; and (b) culturing the host cell in a culture medium to obtain expression of the first, second and third genes.

1 . A method for producing a desired protein (such as a desired heterologous protein) comprising:
 (a) providing a host cell comprising a first recombinant gene encoding a protein comprising the sequence of Orm2p or a variant or fragment thereof and a second gene, such as a second recombinant gene, encoding a desired protein (such as a desired heterologous protein), with the proviso that the first and second genes are not present within the host cell on the same 2 mm-family plasmid; and   (b) culturing the host cell in a culture medium to obtain expression of the first and second genes.   
     
     
         2 . A host cell comprising a first recombinant gene encoding a protein comprising the sequence of Orm2p or a variant or fragment thereof and a second gene, such as a second recombinant gene, encoding a desired protein (such as a desired heterologous protein), with the proviso that the first and second recombinant genes are not present within the host cell on the same 2 mm-family plasmid. 
     
     
         3 . A method according to  claim 1  wherein either or both of the first and second genes are located on a plasmid, and optionally located on the same plasmid. 
     
     
         4 . A method according to  claim 1  wherein the first recombinant gene encoding a protein comprising the sequence of Orm2p or a variant or fragment thereof is located on a plasmid. 
     
     
         5 . A method according to  claim 1  wherein either or both of the first and second genes are integrated into the chromosome of the host cell. 
     
     
         6 . A method according to  claim 1  wherein the first recombinant gene encoding a protein comprising the sequence of Orm2p or a variant or fragment thereof is integrated into the chromosome of the host cell. 
     
     
         7 . A method according to  claim 1  further comprising the step of purifying the thus expressed desired protein (such as a desired heterologous protein) from the cultured host cell or the culture medium. 
     
     
         8 . A method according to  claim 8  further comprising the step of formulating the purified desired protein (such as a desired heterologous protein) with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form. 
     
     
         9 . A plasmid comprising a first recombinant gene encoding a protein comprising the sequence of Orm2p or a variant or fragment thereof and a second gene, such as a second recombinant gene, encoding a desired protein (such as a desired heterologous protein), with the proviso that the plasmid is not a 2 mm-family plasmid. 
     
     
         10 . A method according to any  claim 1  further comprising an additional chaperone protein or variant or fragment thereof, or gene encoding an additional chaperone protein or variant or fragment thereof. 
     
     
         11 . A method according to  claim 11  wherein the additional chaperone is selected from PDI1, JEM1, AHA1, CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8, CNS1, CPR3, CPR6, EPS1, ERO1, EUG1, FMO1, HCH1, HSP10, HSP12, HSP104, HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, MDJ1, MDJ2, MPD1, MPD2, PFD1, ABC1, APJ1, ATP11, ATP12, BTT1, CDC37, CPR7, HSC82, KAR2, LHS1, MGE1, MRS11, NOB1, ECM10, SSA1, SSA2, SSA3, SSA4, SSC1, SSE2, SIL1, SLS1, UBI4, ORM1, ORM2, PER1, PTC2, PSE1, HAC1 or truncated intronless HAC1, TIM9, PAM18 or TCP1 or a variant or fragment of any one of these. 
     
     
         12 . A method according to  claim 1  wherein the desired protein (such as a desired heterologous protein):
 (i) comprises a leader sequence effective to cause secretion from the host cell, such as yeast; and/or 
 (ii) is a eucaryotic protein, or a fragment or variant thereof, optionally a vertebrate or a fungal (such as a yeast) protein; and/or 
 (iii) comprises a sequence selected from albumin, a monoclonal antibody, an etoposide, a serum protein (such as a blood clotting factor, e.g. Factor VII, Factor VIII, Factor IX, Factor X and Factor XIII), antistasin, a tick anticoagulant peptide, transferrin, lactoferrin, endostatin, angiostatin, collagens, immunoglobulins, or immunoglobulin-based molecules or fragment of either (e.g. a dAb, Fab′ fragments, F(ab′) 2 , scAb, scFv or scFv fragment), a Kunitz domain protein, interferons, interleukins, IL10, IL11, IL2, interferon a species and sub-species, interferon b species and sub-species, interferon g species and sub-species, leptin, CNTF, CNTF Ax15 ′, IL1-receptor antagonist, erythropoietin and erythropoietin mimics, thrombopoietin and thrombopoietin mimics, prosaptide, cyanovirin-N, 5-helix, T20 peptide, T1249 peptide, HIV gp41, HIV gp120, urokinase, prourokinase, tPA, hirudin, platelet derived growth factor, parathyroid hormone, proinsulin, insulin, glucagon, glucagon-like peptides, insulin-like growth factor, calcitonin, growth hormone, transforming growth factor b, tumour necrosis factor, G-CSF, GM-CSF, M-CSF, FGF, coagulation factors in both pre and active forms, including but not limited to plasminogen, fibrinogen, thrombin, pre-thrombin, pro-thrombin, von Willebrand's factor, a 1 -antitrypsin, plasminogen activators, nerve growth factor, LACI, platelet-derived endothelial cell growth factor, glucose oxidase, serum cholinesterase, aprotinin, amyloid precursor protein, inter-alpha trypsin inhibitor, antithrombin III, apo-lipoprotein species, Protein C, Protein S, a metabolite, an antibiotic, or a variant or fragment of any of the above. 
 
     
     
         13 . A method according to  claim 13  wherein the desired protein (such as a desired heterologous protein) comprises the sequence of albumin or a variant or fragment thereof. 
     
     
         14 . A method according to  claim 13  wherein the desired protein (such as a desired heterologous protein) comprises the sequence of a transferrin family member, optionally transferrin or lactoferrin, or a variant or fragment thereof. 
     
     
         15 . A method according to  claim 13  wherein the desired protein is a desired heterologous protein that comprises a fusion protein, such as a fusion protein of albumin or a transferrin family member or a variant or fragment of either, fused directly or indirectly to the sequence of another protein. 
     
     
         16 . A method according to  claim 1  wherein the host cell is a eukaryotic cell such as a yeast cell, optionally a member of the  Saccharomyces, Kluyveromyces, Arxula, Yarrowia, Candida, Schizosaccharomyces, Debaryomyces, Xanthophyllomyces, Geotrichum, Ashbya, Hortaea, Schwanniomyces, Trichosporon, Xanthophyllomyces , or  Pichia  genus, such as  Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris, Pichia membranaefaciens, Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomyces fermentati, Kluyveromyces drosphilarum, Pichia methanolica, Hansenula polymorpha  (also known as  Pichia augusta ),  Arxula adeninivorans, Yarrowia lipolytica, Candida boidinii Candida utilis  or  Schizosaccharomyces pombe.    
     
     
         17 . A method according to  claim 1  where, when a 2 mm-family plasmid is used:
 (a) the plasmid is based on pSR1, pSB3 or pSB4 and the host cell is  Zygosaccharomyces rouxii;    
 (b) the plasmid is based on pSB1 or pSB2 and the host cell is  Zygosaccharomyces bailli;    
 (c) the plasmid is based on pSM1 and the host cell is  Zygosaccharomyces fermentati;    
 (d) the plasmid is based on pKD1 and the host cell is  Kluyveromyces drosophilarum;    
 (e) the plasmid is based on pPM1 and the host cell is  Pichia membranaefaciens ; or 
 (f) the plasmid is based on the 2 mm plasmid and the host cell is  Saccharomyces cerevisiae  or  Saccharomyces carlsbergensis.    
 
     
     
         18 . A method according to  claim 1  in which the plasmid is based on the 2 mm plasmid and the host cell is  Saccharomyces cerevisiae  or  Saccharomyces carlsbergensis.