Multicloning vector, expression vector and production of foreign

Details Multicloning vector, expression vector and production of foreign Multicloning vector, expression vector and production of foreign

1995-07-24

1998-10-06

Degen, Nancy

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

4351721, 4352551, 4353201, 536 231, 536 241, 536 242, C12P 2102, C12N 1564, C12N 1581, C07H 2104

Patent

active

058174788

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

The present invention relates to a multicloning vector which enables a fission yeast Schizosaccharomyces pombe (hereinafter referred to as S.pombe) to produce a foreign protein, an expression vector which is obtainable by introducing a gene of a foreign protein into the multicloning vector, a method of preparing the expression vector, a S.Pombe transformant which carries the expression vector, and a method of producing a foreign protein by using the transformant. The present invention enables genes of various foreign proteins to be expressed efficiently and thereby reduces the cost for production of the resulting peptides or proteins.


BACKGROUND ART

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 (inclusive of insect cells) and plant cells. As such foreign proteins, various biogenic polypeptides are considered to be accessible, and many of them have been industrially produced 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. Actually, some products by procaryotic cells are known to be kept away from application to medicines and the like, due to their irregular structures and activities (Proc. Natl. Acad. Sci. USA, 86, 3428-3432, 1989). In addition, Escherichia coli has a characteristic endotoxin, which might contaminate end products.
In methods employing animal or plant cells, production efficiency is low, because these cells are more difficult to handle than microorganisms, their culture is costly, and they are obtainable only at low cell densities. For this reason, the most ideal organisms for production of foreign proteins, especially eucaryotic polypeptides, to be yeasts, eucaryotic microorganism. Yeasts are advantageous in view of their established culture methods and their ability to express genetic information of eucaryotes. Further, these have been conventionally used in fermentation and food industries, are sure to be safe to human bodies, and have a characteristic that they do not contain endotoxins. Among yeasts, S.pombe is considered to be closer to animal cells in properties than Saccharomyces cerevisiae. Therefore, use of S.pombe has 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, such as Japanese Unexamined Patent Publications Nos. 181397/1986, 283288/1990 and 63596/1992. This is because there is no expression vector which has a powerful promoter and can be present stably in S.pombe cells and to which a gene is most suitably and easily introduced.
The present inventors proposed an expression vector capable of resolving the above-mentioned problems (Japanese Unexamined Patent Publication No.15380/1993). However, the expression vector is still insufficient to adequately express genes of foreign proteins, and further improved expression vectors are demanded.


DISCLOSURE OF INVENTION

The present inventors have studies from the above-mentioned aspects, and, as a result, constructed an expression vector that enables the gene of a desired foreign protein to be expressed adequately, by modifying the translation initiation site of the gene of the foreign protein into a restriction enzyme recognition site. The

REFERENCES:
Whitehead et al. "Three Restriction Endonucleases from Anabaena-flos-Aquae" J Gen Microbiol 131 951-858 1985.

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