Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-08-01
2003-10-28
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S254300, C435S233000
Reexamination Certificate
active
06638737
ABSTRACT:
The present invention relates to a novel enzyme. In particular, the invention relates to a novel cyclophilin-like peptidyl prolyl cis-trans isomerase.
In most protein over-production strategies, strong promoters capable of directing very high levels of transcription are used to over-express genes encoding heterologous and homologous proteins. Limitations in protein secretion are likely to be due to bottlenecks at the translational and post-translational levels (Tsuchiya, K. et al., (1992) Applied Microbiology and Biotechnology 38:109-114). Proper folding of the protein is required for export competence. Overexpressed heterologous proteins may fold improperly and then be degraded during protein traffic through the secretory pathway. Therefore, the correction of folding defects is desirable in order to increase protein secretion.
The folding of a protein is catalysed by an number of factors, including two isomerase families, namely protein disulphide isomerase, catalysing disulphide bond formation, and peptidyl prolyl cis-trans isomerase, catalysing the isomerisation of Xaa-Proline bonds.
Overexpression of protein disulphide isomerase in
S. cerevisiae
results in an increase in secretion of human platelet-derived growth factor (Robinson et al., 1994). However, the overproduction of
A. niger
protein disulphide isomerase did not result in an increase in secretion of hen egg white lysozyme (HEWL) or glucoamylase (Ngiam C., Jeenes, D. J. J., Punt, P. J., van den Hondel, C. A. M. J. J., Archer, D. A. (1998); Characterisation of a foldase, PDIA, in the protein secretory pathway of
Aspergillus niger
; submitted.).
One of the slowest steps in protein folding is the cis-trans isomerisation of Xaa-proline bonds. This isomerisation is markedly accelerated when peptidyl prolyl cis-trans isomerases are present. Peptidyl prolyl cis-trans isomerases of the cyclophilin family from different organisms have been shown to possess foldase activity in vitro (Schönbrunner E. R., Mayer S., Tropschug M., Fischer G., Takahashi N., Schmid, F. (1991); Catalysis of protein folding by cyclophilins from different species. J Biol Chem 266: 3630-3635). These isomerases are inhibited by the immuno-suppressant drug cyclosporin A. The effects on secretion of heterologous peptides by overexpression of peptidyl prolyl cis-trans isomerases are however not known in the prior art.
Proteins active in the E.R. are targeted to this compartment by a carboxy terminal extension of 4 amino acids. In
A. niger
HDEL (SEQ ID NO: 7) and KDEL (SEQ ID NO: 9) have been reported to function as an E.R. retention signal (Jeenes D. J., et al., (1997) Gene 193:151-156).
SUMMARY OF THE INVENTION
In a first aspect of the present invention, there is provided a method for producing a secretable polypeptide in an host cell, comprising overexpressing a peptidyl prolyl cis-trans isomerase in the cell, thereby increasing the yield of the secreted polypeptide.
In a second aspect, the invention relates to a polypeptide possessing foldase activity characterised by having a capability to catalyse the cis-trans isomerisation of a peptide bond on the N terminal side of proline residues in polypeptides, having a signal sequence at the N-terminus and an endoplasmic reticulum retention signal at the C-terminus, and a molecular weight of 20.7 kDa and a deduced isoelectric point of 6.27.
In a third aspect, the invention relates to a polypeptide possessing foldase activity characterised by having a capability to catalyse the cis-trans isomerisation of a peptide bond on the N terminal side of proline residues in polypeptides, encoded by a nucleic acid capable of hybridising under conditions of low, medium or high stringency with a 17 base oligonucleotide derived from SEQ ID No. 1.
In a fourth aspect, the invention relates to a polypeptide possessing foldase activity characterised by having a capability to catalyse the cis-trans isomerisation of a peptide bond on the N terminal side of proline residues in polypeptides, encoded by a nucleic acid capable of hybridising under conditions of low, medium or high stringency with a 20 base oligonucleotide derived from SEQ ID No. 2.
In a fifth aspect, the invention relates to a polypeptide possessing foldase activity characterised by having a capability to catalyse the cis-trans isomerisation of a peptide bond on the N terminal side of proline residues in polypeptides, which is at least 40% homologous to SEQ. ID. No. 2.
REFERENCES:
patent: 6127148 (2000-10-01), Carlow et al.
patent: 2001/0034045 (2001-10-01), Penttila et al.
patent: 0 413 440 (1991-02-01), None
patent: 0 455 316 (1991-11-01), None
patent: WO 94/23040 (1994-10-01), None
patent: WO 94/24292 (1994-10-01), None
patent: WO 95/10616 (1995-04-01), None
patent: WO 96/01323 (1996-01-01), None
patent: WO 96/29416 (1996-09-01), None
Drkx, P.M.F., et al. (2001) Mol. Genetics & Genomics, pages unknown, published online.*
Xu et al,Science in China, 38(4):429-437 (1995).
Chen et al, “A cyclophilin from the polycentric anaerobic rumen fungus Orpinomyces sp. Strain PC-2 is highly Homologous to vertebrate cyclophilin B”, Proc. Natl. Acad. Sci. USA, vol. 92, No. 7, pp. 2587-2591 1995, Biochemistry, Natl Academy of Science, Washington, XP002129590.
Hornbogen et al, “Two new cyclophilins from Fusarium sambucinum and Aspergillus niger”, Biochem. & Biophys. Research Comm., vol. 187, No. 2, pp. 791-796, 1992, Academic Press, Inc.
Tsuchiya et al., “High level expression of the synthetic human lysozyme gene inAspergillus oryzae,”Applied Microbiology and Biotechnology(1992), vol. 38, pp. 109-114.
Schonbrunner et al., “Catalysis of protein folding by cyclophilins from different species,”J. Biol. Chem.(1991), vol. 266, No. 6, pp. 3630-3635, The American Society for Biochemistry and Molecular Biology, Inc..
Jeenes et al., “Isolation and characterisation of a novel stress-inducible PDI-family gene fromAspergillus niger,” Gene(1997), vol. 193, pp. 151-156.
Bergsma et al., “The Cyclophilin Multigene Family of Peptidyl-Prolyl Isomerases,”J. Biol. Chem.(1991), vol. 266, No. 34, pp. 23204-23214, the American Society for Biochemistry and Molecular Biology, Inc.
Rouviere et al., “SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins,”Genes&Dev.(1996), vol. 10, pp. 3170-3182, Cold Spring Harbor Laboratory Press, ISSN: 0890-9369..
Wang et al., “Enzymes as chaperones and chaperones as enzymes,”FEBS lett.(1998), vol. 425, pp. 382-382, Federation of European Biochemical Societies.
Gurr et al, “The Structure and organization nuclear genes of filamentous fungi,”Gene Structure in Eukaryotic Microbes, (1987), pp. 93-139, IRL Press, Oxford.
Rambosek et al., “Recombinant DNA in filamentous fungi: Progress and Prospects”CRC Crit. Rev. Biotechnol.(1987), vol. 6, Issue 4, pp. 357-393.
Davies, “Heterologous gene expression and protein secretion in Aspergillus” In: Martinelli et al., Aspergillus:50 years on—Progress in industrial microbiology, (1994), vol. 29, pp. 527-560, Elsevier Amsterdam.
Ballance, “Transformation systems for filamentous funcgi and an Overview for Fungal Gene structure,” In Leong et al. (editors)Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi(1991), pp. 1-29, Marcel Dekker Inc. New York.
Turner, “Vectors for genetic manipulation” In Martinelli et al. (editors) Aspergillus:50 years on: Progress in industrial microbiology(1994), vol. 29, pp. 641-666, Elsevier Amsterdam.
Broekhuijsen et al., “Secretion of heterologous proteins by Aspergillus niger: Production of active human interleukin-6 in a protease-deficient mutant by KEX2-like preocessing of a glucoamylase-hlL6 fusion protein,”J. Biotechnol(1993), vol. 31, pp. 135-145, Elsevier Science Publishers B.V..
Myers et al. “Optimal Alignments in Linear Space”CABIOS(1988), vol. 4, No. 1, pp. 11-17, IRL Press Limited, Oxford, England.
Wilbur et al., “Rapid similarity searches of nucleic and acid protein data banks,”Proc. Natl. Acad. Sci. USA(1983) vol. 80, pp. 726-730.
Needleman et al. “A general method applicable to the search for sim
Derkx Patrick M. F.
Madrid Susan M.
Danisco A/S
Foley & Lardner
Patterson Jr. Charles L.
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