Secretion signal gene and expression vector containing it

Details Secretion signal gene and expression vector containing it Secretion signal gene and expression vector containing it

1996-10-02

1999-07-06

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...

4352523, 43525411, 4353201, 536 231, 536 241, C12N 1500

Patent

active

059196548

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

The present invention relates to a secretion signal gene having a base sequence encoding a polypeptide derived from the secretion signal of a precursor of a mating pheromone (P-factor), which concerns mating of fission yeast Schizosaccharomyces pombe (hereinafter referred to as S. pombe). The present invention also relates to an expression vector containing the secretion signal gene and a foreign protein structural gene, and efficient secretory production of the foreign protein structural gene product by S. pombe or the like.


BACKGROUND ART

Hitherto, production of foreign proteins utilizing genetic recombination technology has been extensively conducted by using microorganisms such as Escherichia coli, Saccharomyces cerevisiae or Bacillus, animal cells, plant cells and insect cells. As such foreign proteins, various biogenic proteins are considered to be accessible, and many of them have been industrially produced by using these living organisms for medical use so far.
However, methods employing procaryotes are not effective for all polypeptides, and it is not always easy to reproduce the complicated post-translational modification of eucaryotic proteins and to reproduce the natural steric structures. In addition, Escherichia coli has a characteristic endotoxin, which might contaminate end products. On the other hand, as for methods employing animal, plant or insect cells, these cells are more difficult to handle than microorganisms, their culture is costly, and production efficiency is low. For this reason, yeasts, eucaryotic microorganisms, are considered as the best for production of foreign proteins, especially eucaryotic proteins. Their culture methods are well established, and they do not contain endotoxins. Therefore, expression vectors for use in various yeast hosts have been developed so far (Romanos, M. A. et al., Yeast 8, 423-488, 1992).
Among various yeasts, S. pombe is considered to be closer to higher animals in various properties such as cell cycle, chromosomal structure and RNA splicing than other yeasts inclusive of Saccharomyces cerevisiae. The post-translational modification such as acetylation, phosphorylation and glycosylation of proteins produced in S. pombe seems fairly similar to that in animal cells (Russell, P. R. and Nurse, P., Cell 45, 781-782, 1986; Kaufer, N. F. et al, Nature 318, 78-80, 1985; Chappell, T. G. and Warren, G., J. Cell. Biol. 109, 2693-2702, 1989). Therefore, use of S. pombe as a host for expression of a foreign protein is expected to provide a gene product closer to its natural form, like that produced by animal cells. Since yeasts have a lot of commonness in their culture methods, knowledges about other yeasts can be easily applied to the yeast. Therefore, it is obviously advantageous to use S. pombe for production of a foreign protein by using microbiological methods and the DNA recombination technique.
However, S. pombe is far behind Escherichia coli and Saccharomyces cerevisiae in studies on genetic recombination using them. Especially, with respect to gene expression in S. pombe, only a small number of studies have been reported (Japanese Unexamined Patent Publications Nos. 181397/1986, 283288/1990 and 63596/1992). This is because development of expression vectors which have powerful promoters, are stable in S. pombe cells and are suitable and convenient for introduction of a gene has been retarded. Recent development of vectors for the fission yeast with a high expressivity which contains an animal virus-derived promoter region eventually opened the way to mass production of foreign proteins by S. pombe (Japanese Unexamined Patent Publications Nos. 15380/1993 and 163373/1995, which disclose inventions of the present inventors). This technique enabled many intracellular proteins to be produced easily and therefore is fairly useful.
Production of (foreign) eucaryotic secretory proteins by yeasts scarcely succeeded so far because yeasts can hardly recognize inherent signal sequences of foreign secretory proteins and therefore can not secre

REFERENCES:
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patent: 5712113 (1998-01-01), Chung et al.
von Heijne (1986) Nucleic Acids Res., vol. 14, No. 11, pp. 4683-4690, 1986.
Imai et al. (1994) Genes and Development, vol. 8, pp. 328-338, 1994.
Tohda et al. (1994) Gene, vol. 150, pp. 275-280, 1994.

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