Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1994-06-07
1998-01-06
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, C12N 1562, C12P 2102
Patent
active
057053588
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The invention relates to a process for the production and optionally secretion of a protein by means of a transformed mould, into which an expression vector has been introduced with the aid of recombinant DNA techniques known per se, said vector comprising one or more mould-derived expression and/or secretion regulating regions controlling the expression of a gene encoding said protein and optionally controlling the secretion of the protein so produced. Such a process is known from various publications, in which the production of proteins with the aid of transformed moulds is described. Thus, in the non-prior-published patent application PCT/EP 91/01135 (UNILEVER, in the priority year published on 26 Dec. 1991 as WO 91/19782) there is described, inter alia, the production of a homologous endoxylanase II protein by a transformed Aspergillus strain.
Other ways of producing proteins by transformed moulds, in particular while using promoters originating from Aspergillus moulds, are known. transformed Aspergillus niger var. awamori of the milk-clotting enzyme chymosin or its precursor prochymosin. It was concluded that production of a fusion protein in which the prochymosin was connected with its N-terminus to the C-terminus of the Aspergillus protein glucoamylase gave a much higher secretion than with production of the prochymosin alone, whereby in both cases the protein was preceded by the glucoamylase signal sequence and under control of the glucoamylase promoter. construct--containing promoter for use in Aspergillus", published on 1 Mar. 1991, the constitutive promoter of the Aspergillus nidulans aldehyde dehydrogenase gene and its use for the production of heterologous proteins in a transformed mould is described. dehydrogenase (GAPDH) gene--derived from Aspergillus orizae, used in new expression system in yellow-green or black koji mould", published on 17 Jul. 1991, the use of the promoter and terminator of the GAPDH gene in a vector for transforming a mould to produce foreign proteins is described. construct vectors for expression of structural genes in suitable hosts", published on 7 Aug. 1991, the overproduction of a homologous gene product or a heterologous gene product in A. niger is described.
Moulds are organisms frequently used in the production of proteins and metabolites. A biotechnologically very important aspect of moulds is that they are capable of very efficient protein production and, if desired, secretion into the medium. It is also possible to grow moulds in a properly controlled way in large bioreactors. The combination of the possibilities of generating fungal biomass cost-effectively by means of fermentation and the high specific expression per cell make moulds exceptionally interesting hosts for the production of both heterologous and homologous proteins. For efficient production of these heterologous and homologous proteins, the use of an efficient promoter effective in moulds is essential. For secretion of a protein into the medium, specific sequences are required that cater for this. In connection with possible toxicity for the mould cell of the protein to be produced, it is also important that the activity of a promoter can be regulated, i.e. turned on at suitable moments, thus an inducible promoter is preferred.
SUMMARY OF THE INVENTION
The invention is based on the use of a non-prior-published promoter, which is described in more detail below, as well as on the use of other expression and/or secretion regulating regions, such as a terminator, a DNA sequence encoding a signal sequence, and a DNA sequence encoding at least an essential part of a mature endogenous mould protein.
In studies of the expression of proteins in moulds it was found that the enzyme endoxylanase type II (exlA) was efficiently produced after induction of expression of the exlA gene, and was also secreted efficiently into the medium. For production of that protein, the encoding gene was cloned together with its own promoter. In comparison with other mould promoters, the endoxyl
REFERENCES:
Ward, et al.: "Improved Production of Chymosin in Aspergillus by Expression as a Glucoamylase-chymosin Fusion", Bio/Technology, vol. 8, No. 5, May 1990, pp. 435-440.
WO,A,9 119 782--Dec. 26, 1991--see p. 7, line 32--p. 8, lien 7; figue 1--see p. 10, line 34, p. 11, line 12.
N. Roberts, R.P. Oliver, P.J. Punt and C.A.M.J.J. van den Hondel, Curr Genet, Expression of the Escherichia coli .beta.-glucuronidase gene in Industrial and Phytopathogenic Filamentous Fungi Curr Genet (1989), 15:177-180.
Fuller, Enzymers Required for Yeast Prohormone Processing, Ann. Rev. Physiol., 1988, 50:345-62.
Gunnar von Heijne, A New Method for Predicting Signal Sequence Cleavage Site, Nucleic Acids Research, 1986, vol. 14, No. 11, pp. 4683-4690.
Sanger et al., DNA Sequencing with Chain-terminating Inhibitors, Proc. natl. Acad. Sci. USA, Dec. 1977, vol. 74, No. 12, pp. 5463-5467.
Gouka Robertus Johannes
Musters Wouter
Stam Hein
van den Hondel Cornelis Antonius
Verbakel Johannes Maria
Bugaisky Gabriele E.
Unilever Patent Holdings B.V.
Wax Robert A.
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