Method of making proteins in transformed yeast cells

Details Method of making proteins in transformed yeast cells Method of making proteins in transformed yeast cells

1999-07-06

2002-03-19

McKelvey, Terry (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...

C435S091420, C435S483000

Reexamination Certificate

active

06358705

ABSTRACT:

FIELD OF INVENTION
The present invention relates to expression of proteins in transformed yeast cells, DNA construct and vectors for use in such process and yeast cells transformed with the vectors.
BACKGROUND OF THE INVENTION
It is well known to use transformed yeast strains for the expression of proteins see for example European patent applications Nos. 0088632A, 0116201A, 0123294A, 0123544A, 0163529A, 0123289A, 0100561A, 0189998A and 0195986A, PCT patent applications Nos. WO 95/01421, 95/02059 and WO 90/10075, and U.S. Pat. No. 4,546,082.
It is a common feature of the above methods that the yeast production plasmid contains a gene for an antibiotic marker. Such marker gene stems from the initial cloning steps in
E. coli
where it was used to screen for transformed cells or to maintain plasmids used as vectors. The antibiotic marker genes are not believed to have any adverse impact on the culturing of the transformed yeast cell and it has therefore been common practice not to take any steps to delete such DNA. In addition, characterization of the plasmid construct is usually done by isolation of plasmids from the transformed yeast cells and transformation of the isolated plasmid into
E. coli
followed by antibiotic selection. It has thus been convenient for practical purposes to retain the antibiotic resistance marker gene.
Although both research laboratories and industrial production plants are controlled by very severe safety regulations there is always a small risk that a few cells by accident will be released to the environment. Due to their highly sophisticated nature such genetically engineered microorganisms will only survive for a very short period and the risk of harming the environment is extremely low. This is of course the reason why such transformed microorganisms have been approved for use in both research and in large scale operations.
Even if the cells die quickly the plasmids containing the antibiotic resistant gene may still accidentally be disposed to the environment and there is a theoretical risk of introduction of resistance to antibiotics in bacteria if the plasmid is taken up spontaneously.
Antibiotics are of great importance for treatment of human and animal bacterial infections. Any risk of a potential environmental contamination with a gene that confers resistance to antibiotics should be minimized, if possible.
There is therefore a need to develop even more safer methods than the methods used up to this day and it is the object of the present invention to provide such improved methods.
SUMMARY OF THE INVENTION
The present invention relates to a method for expressing heterologous proteins or polypeptides in yeast wherein the yeast transformant strain used for production contains an expression vector in which an antibiotic marker gene used in the initial cloning steps has been made non functional by in vitro modification before transformation of the yeast host. The present invention also relates to DNA sequences and expression vectors for use in such method and to transformed yeast cells.
According to one aspect the present invention is related to a recombinant yeast expression vector being unable to confer antibiotic resistance to bacteria cells and comprising a gene coding for a heterologous gene and an antibiotic resistance marker gene which has been made non functional by in vitro modification.
According to a further aspect the present invention is related to a method for making a desired polypeptide or protein said method comprises culturing a yeast strain comprising a vector being unable to confer antibiotic resistance to bacteria cells and comprising a gene coding for a heterologous gene and an antibiotic resistance marker gene, which marker gene has been made non functional by in vitro modification before transformation of the yeast host, and isolating the desired product from the culture medium.
The method according to the invention will typically comprise culturing a yeast strain containing a yeast expression plasmid in which plasmid a functional antibiotic marker gene used for initial cloning steps in bacteria has been made non-functional by in vitro deletion of part of the marker gene or the whole marker gene before insertion into the yeast host to be used for expression and secretion of the desired polypeptide or protein.
The deletion of the antibiotic marker gene is preferably made by insertion of suitable restriction cleavage sites on each side of the antibiotic resistance marker gene whereupon the marker gene is deleted by in vitro treatment with suitable restriction enzymes.
The present invention is also related to transformed yeast strains comprising a vector being unable to confer antibiotic resistance to bacteria cells and comprising a gene coding for a heterologous gene and an antibiotic resistance marker gene which has been made non functional by in vitro modification before transformation of the yeast host.
The yeast strain is preferable a Saccharomyces strain, and in particular a
Saccharomyces cerevisiae
strain.
As used herein the expression “antibiotic marker gene” or “antibiotic resistance marker gene” means a gene that allows phenotypic selection of transformed bacterial cells and plasmid amplification.
The most commonly used antibiotic resistance marker genes in
E. coli
are the ampicillin (AMP), chloramphenicol, neomycin, kanamycin and tetracylin resistance conferring marker genes.
As used herein the expression “non functional marker gene” means that the marker gene has been either deleted or made non functional by deletion of part of the gene. It is preferred that the gene is completely deleted.
As used herein “in vitro modification” means modification steps performed on the vector outside the cell environment.
As used herein “unable to confer antibiotic resistance to bacteria cells” means that the antibiotic resistance genes are non functional in any organism due to the described gene manipulation.
As used herein the “yeast host” means a yeast organism to be transformed or transfected with the expression plasmid or vector.


REFERENCES:
patent: 0 241 435 (1987-10-01), None
patent: 0 251 579 (1988-01-01), None
patent: 0 284 044 (1988-09-01), None
patent: 0 286 424 (1988-10-01), None
patent: 0 406 003 (1991-01-01), None
patent: 0 438 200 (1991-07-01), None
patent: 0 501 914 (1992-09-01), None
patent: 0 592 358 (1994-04-01), None
patent: 0 635 574 (1995-01-01), None
patent: 0 674 006 (1995-09-01), None
patent: 0 814 165 (1997-12-01), None
patent: WO 94/00554 (1994-01-01), None

Affiliated with

Also associated with

Rating

Method of making proteins in transformed yeast cells does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method of making proteins in transformed yeast cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of making proteins in transformed yeast cells will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2842020