Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-03-01
1998-04-21
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...
4352542, 435 711, 435 697, 43525411, 536 231, 530350, C12P 2100
Patent
active
057416745
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a process for the recombinant production of proteins in the yeast Hansenula.
The recombinant production of proteins in the yeast Hansenula is known. European Patent 173378 describes the recombinant preparation of proteins using particular promoter elements of MOX or DAS. However, this document provides no information as to how efficient secretion and correct processing of the required protein is to be achieved.
Furthermore, it is known that in Hansenula polymorpha, the glucoamylase leader sequence (GAM1) from Schwanniomyces occidentalis is recognized as signal sequence, and it is possible to secrete correctly processed glucoamylase (G. Gellissen et al., Bio-technology 9 (1991) 291-295). However, this signal sequence does not lead to the secretion of gene products foreign to yeasts, for example it is not possible to secrete the protein hirudin therewith.
It is an object of the present invention to provide a process for the recombinant production of proteins, in particular of proteins which are foreign to yeasts, ie. heterologous, in the yeast Hansenula, which ensures efficient secretion and correct processing for a large number of proteins.
We have found that this object is achieved by a process for the recombinant production of proteins in the yeast Hansenula, which comprises transforming Hansenula with an expression cassette which comprises the following structural elements encoded:
It is possible to use as leader sequence L the leader sequences of all gene products secreted in yeast, which are recognized by Hansenula. It is not a necessary requirement that the leader sequence originates from a Hansenula gene. Leader sequences of yeasts of genera other than Hansenula are also suitable, for example Saccharomyces or Schwanniomyces. A leader sequence which is very suitable for the invention is, for example, the alpha factor leader sequence from Saccharomyces cerevisiae (MAT.alpha.).
Leader sequences which are preferably used are those of strongly expressed and secreted hydrolytic enzymes such as alpha-amylase, invertase, acid phosphatase or glucoamylase. The glucoamylase leader sequence from Schwanniomyces occidentalis is particularly preferably used.
Suitable sequences as adaptor A are all those which code for a polypeptide which contains an alpha-helix structure. The presence of an alpha-helix structure can be determined by the algorithm of Garnier et al. (J. Mol. Biol. 120 (1978) 97-120). It is particularly easy to determine, using commercially obtainable computer programs based on this algorithm, whether a polypeptide sequence ought to have an alpha-helix structure.
As a rule, sequences which are very suitable as adaptor are all those for which the computer program Microgenie.RTM. (Beckmann) calculates for ALPHA a larger positive value than for the three other possible structures (BETA, TURN, COIL) for a peptide sequence of at least four amino acids in the region of the processing site A-P-GEN.
The length of the adaptor sequence A can vary within wide limits for the use according to the invention. As a rule, it is from five to one hundred amino acids.
A sequence of the glucoamylase from Schwanniomyces occidentalis which contains amino acids 23-72 (GAM 23-72; Dohmen et al. Gene 95 (1990), 111-121) is preferably used as adaptor sequence.
This sequence can be used as adaptor sequence directly or, particularly preferably, after extension at the C terminus by one to four amino acids. Parts of this sequence, preferably those obtained by N-terminal truncation, are also very suitable for the process according to the invention.
It is also possible, for example, by means of the computer program described above, for the sequence regions which particularly contribute to the alpha-helix formation to be identified and also optimized in respect of the alpha-helix structure by exchange of individual amino acids.
A sequence which has proven particularly suitable as adaptor for the preparation of thrombin inhibitors, especially hirudin and hirudin derivatives, by the process according to t
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Winther et al. (1991) Proc. Natl. Acad. Sci. USA, vol. 88, pp. 9330-9334, 1991.
Hiramatsu et al. (1991) Applied Environmental Microbiology, vol. 57, pp. 2052-2056, 1991.
Sudbery et al. (1988) Biochemical Society Transactions, vol. 16, pp. 1081-1083, 1988.
Chem. Abst., vol. 116, 1992 -116: 10032 p. 174.
Biotech., vol. 9, Mar. 1991, Heterologous Gene Expression . . . 291-295.
Agric. Bio. Chem., 53 (2), 483-489, 1989, Threonine-and Serine-rich Tract of the . . .
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Bollschweiler Claus
Janowicz Zbigniew A.
Piontek Michael
Schweden Jurgen
Strasser Alexander W.M.
Longton Enrique D.
Rhein Biotech Gesellschaft fur neue biotechnologische Prozesse u
Wax Robert A.
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