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
2000-02-29
2001-05-22
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S254200, C435S477000, C536S023100
Reexamination Certificate
active
06235499
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for transforming the fission yeast
Schizosaccharomyces pombe
, particularly a method for transfer of a heterologous protein structural gene to its chromosome through homologous recombination. The present invention also relates to a vector for use in the transformation method, a
Schizosaccharomyces pombe
transformant obtained by the method and a method for producing a heterologous protein by using the
Schizosaccharomyces pombe
transformant.
2. Discussion of Background
The gene recombination technology is applied in various industries for production of heterologous proteins by usually using
Escherichia coli
, the budding yeast
Saccharomyces cerevisiae,
the methanol-assimilative yeast
Pichia pastoris,
insect cells and animal cells as the hosts. Ideally, any natural and artificial proteins could be produced, and in recent years, attempts have been made to produce not only purified proteins but also various chemical substances.
However, no “general host” that enables efficient production of any protein or chemical substance has been developed yet, and individual production systems have been developed for different target proteins and chemical substances by trial and error. Therefore, a further technical breakthrough is demanded in each expression system.
For production of heterologous proteins, especially eucaryotic proteins, use of eucaryotic microorganisms is considered as the best approach. Yeasts are very popular due to its long intimate relation with human life as a foodstuff in human history, have established large scale culture methods and have no endogenous substances detrimental to the human body unlike other expression systems. Various expression systems using yeasts as hosts have been developed so far (Yeast, 8, 423 (1992)).
In particular, a fission yeast,
Schizosaccharomyces pombe
(hereinafter referred to as
S. pombe
) is said to be closer to animal cells than other yeasts inclusive of a budding yeast
Saccharomyces cerevisiae
in various properties such as cell cycle, chromosomal structure and RNA splicing, and the post-translational modification such as acetylation, phosphorylation and glycosilation of proteins produced in
S. pombe
seems similar to that in animal cells (Cell, 45, 781 (1986); Nature, 318, 78 (1985); J. Cell. Boil., 109, 2693 (1989)).
Besides, despite being a eucaryote,
S. pombe
has been studied extensively for its high versatility in genetics, molecular biology and cell biology as a unicellular organism (Molecular biology of the fission yeast, Academic Press (1989)). Technological studies have been already done for DNA recombination in
S. pombe
(Experiments with Fission Yeast, Cold Spring Harbor Laboratory press (1993)).
For these reasons,
Schizosaccharomyces pombe
is considered advantageous as the host for expression of inherent proteins in animal cells. Use of
Schizosaccharomyces pombe
is expected to provide a gene product closer to its natural form in animal cells. However,
Schizosaccharomyces pombe
is far behind the budding yeast in studies on gene expression, and the literature on gene expression using
Schizosaccharomyces pombe
has been scant (JP-A-61-181397, JP-A-2-283288, JP-A-4-63596). 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 high-expressivity vectors for the fission yeast such as an inducible expression vector using the promoter region for the nmt1
+
gene (pREP1) and vectors having an animal virus-derived promoter region eventually opened the way to large scale production of heterologous proteins in
S. pombe
(JP-A-5-15380, JP-A-7-163373, WO96/23890, JP-A-10-234375).
These technologies have facilitated production of many intracellular proteins and provided useful expression systems. In fact,
Schizosaccharomyces pombe
has gradually come into wide use as a host for expression of heterologous protein genes and is known to be suited especially to expression of genes from animal cells including human cells (Foreign gene expression in fission yeast
Schizosaccharomyces pombe
, R. G. Landes (1997)). For its advanced membrane structures including the Goldi body and the endoplasmic reticulum,
Schizosaccharomyces pombe
is also used for expression of membrane proteins and shows high level expression.
A conventional
S. pombe
transformant carries the expression vector introduced therein as an extrachromosomal genetic material (a plasmid). However, the extrachromosomal genetic material sometimes slips out and disappears from
S. pombe
cells during incubation. Therefore, in order to stably maintain a vector having a heterologous protein structural gene in cells, it is preferable to integrate the vector into a chromosome of
S. pombe.
Chromosomal integration of vectors usually involves homologous recombination. A vector capable of being integrated into a chromosome through homologous recombination will be referred to as an integrative vector hereinafter.
Integration of integrative vectors into chromosomes of
S. pombe
has been reported already (J. Ind. Microbiol., 4, 409 (1989); Appl. Microbiol. Biotechnol., 49, 45 (1998)). However, conventional integrative vectors can not provide
S. pombe
transformants which show satisfactory expression of heterologous proteins.
The present inventors investigated the reasons and have found that one of the reasons is the low efficiency in chromosomal integration of integrative vectors into
S. pombe
attributable to many unintegrated vector molecules present as plasmids in
S. pombe
cells. On the other hand, it is generally known that more than one copy of an integrative vector can be integrated at one site in a chromosomal gene, and the expression of a heterologous protein is expected to increase with the number of copies of an integrative vector integrated at one gene site. The present inventors found that conventionally obtainable
S. pombe
transformants have a few copies of an integrative vector at one site in a chromosome and speculated that this might be the reason of the unsatisfactory expression of heterologous proteins. The present inventors have conducted extensive research into the reasons of the above-mentioned low efficiency in chromosomal integration of integrative vectors into
S. pombe
and the insufficient number of copies of an integrative vector integrated at one gene site, and as a result, have found that the “replication origin in an integral vector which functions in cells other than
S. pombe
cells required for construction of the vector” is responsible for them.
Construction of a vector such as an integrative vector essentially involves replication of the vector by using cells which are easy to genetically engineer.
Escherichia coli
is a typical example of such cells and frequently used for construction of vectors. The presence of the replication origin from
Escherichia coli
called “ori” in a vector permits replication of the vector in
Escherichia coli.
The present inventors have found that the presence of the replication origin lowers the efficiency in chromosomal integration of integrative vectors into
S. pombe
and decrease the number of integrated copies.
On the basis of the above-mentioned discoveries, the present inventors have found that the above-mentioned problem is solved by chromosomal integration of an integrative vector into
S. pombe
after removal of the above-mentioned replication origin, namely that an integrative vector which has lost the above-mentioned replication origin is integrated with improved efficiency and increases the number of copies integrated at one site of a chromosomal gene.
The present invention provides the following transformation methods, vector, transformant and method for protein production using the transformant:
a method for transforming
Schizosaccharomyces pombe
which comprises integrating a vector into a chromosome of
Schizosaccharomyces p
Hama Yuko
Tohda Hideki
Asahi Glass Company Limited
Davis Katherine F
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Schwartzman Robert A.
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