Cold-inducible expression vector

Details Cold-inducible expression vector Cold-inducible expression vector

2000-05-19

2002-11-12

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

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

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

C435S320100, C536S023100

Reexamination Certificate

active

06479260

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to vector used in a recombinant DNA technique and also to a method for the expression of protein using the said gene.
PRIOR ART
Production of useful proteins using a recombinant DNA technique is a widely-used art at present. Among that, an expression system using
Escherichia coli
as a host is the most commonly used expression system and many proteins have been produced by means of recombinants. It is common for the production of useful proteins by such recombinants to use the so-called expression vector which is constructed by arranging the desired genes under the control of promoter recognized by RNA polymerase. Examples of the promoter used for expression vector are lac, trp, tac, gal and ara when
E. coli,
for example, is used as a host. An example of expression vector utilizing a promoter which is other than that directly recognized by RNA polymerase of
E. coli
is a pET-system (manufactured by Novagen) [
J. Mol. Biol.,
volume 189, pages 113-130 (1986);
Gene,
volume 56, pages 125-135 (1987)] which utilizes a promoter recognized by RNA polymerase of bacteriophage T7 infecting to
E. coli.
In the case of the pET-system, T7RNA polymerase is expressed in
E. coli,
transcription of the desired gene which is arranged at the downstream of T7 promoter on the expression vector by the said T7RNA polymerase takes place and synthesis of the desired protein occurs by means of a translation system of the host.
However, when the desired protein is expressed in a high level in many
E. coli
expression system including the pET-system, it often happens that the desired protein gives an insoluble complex (the so-called inclusion body) and the amount of the desired protein of an active type becomes very small. It has been reported that, in some polypeptides, there are examples where the inclusion body is solubilized and then subjected to a refolding operation to give a polypeptide of an active type. Usually, however, its recovered amount is often low and, in addition, it is necessary to investigate an appropriate refolding condition for each of the desired proteins. Therefore, there has been a demand for a system where the protein of an active type is directly expressed in
E. coli.
Formation of an inclusion body is believed to take place in such a manner that, during the stage of an intermediate before the translated polypeptide chain is folded to a correct steric configuration, interwinding with other polypeptides takes place due an intermolecular action whereupon a very big insoluble complex is formed. It has been known that, when incubation of a recombinant
E. coli
is carried out at the temperature (20-30° C.) which is lower than the commonly-used temperature of 37° C. in that case, the expressed amount of the protein of an active type increases. That is presumed to be due to the fact that sufficient time is available for folding of the intermediate to a correct structure because of retardation of a translation rate by ribosome and that stability of the expressed protein of an active type increases because of retardation of action of the intracellular protease under a low temperature condition. As such, in the production of protein which is apt to give an inclusion body, a method where a recombinant
E. coli
is incubated at low temperature has been receiving public attention as an effective means.
On the other hand, when the incubating temperature of
E. coli
during a logarithmic growth period is lowered form 37° C. to 10~20° C., growth of
E. coli
once stops and, during that period, expression of a group of proteins called cold-shock proteins is induced. The said proteins are classified into group I (10-fold or higher) and group II (lower than 10-fold) depending upon their inducing levels and examples of the proteins of the group I are CspA, CspB, CspG and CsdA. In the case of CspA among them, its expressed amount after 1.5 hours from the temperature shift of from 37° C. to 10° C. reaches 13% of the total cell protein [
Proc. Natl. Acad. Sci. USA,
volume 87, pages 283-287 (1990)] and, therefore, utilization of promoter of cspA gene for the production of recombinant protein at low temperature has been attempted.
In addition to the above-mentioned advantage of initiation of transcription in a high efficiency at low temperature by a promoter of cspA gene, the following effectiveness has been shown for the recombinant protein expression system under a low temperature condition using the cspA gene.
(1) When a translatable mRNA transcribed from cspA gene does not code for CspA protein having a function or, to be more specific, when it codes for only a part of N-terminal sequence of CspA protein, such an mRNA inhibits the expression of other
E. coli
proteins including the cold-shock protein for long time and, during that period, translation of the said mRNA is carried out preferentially [
J. Bacteriol.,
volume 178, pages 4919-4925 (1996)].
(2) In a position at a 12-base downstream from an initiation codon of cspA gene, there is a sequence called a downstream box consisting of 15 bases and it increases the translation efficiency under a low temperature condition.
(3) A 5′-untranslated region consisting of 159 bases existing at from the initiation point of transcription of cspA gene mRNA to the initiation codon gives a negative influence and a positive influence on the expression of CspA at 37° C. and at low temperature, respectively.
However, although the promoter of the said gene is surely able to initiate the transcription at low temperature in a high efficiency, it actually acts even at the common incubating temperature (37° C.) and it has been suggested that the stability of mRNA transcribed from the said gene regulates the expression of the said gene as well [
Molecular Microbiology,
volume 23, pages 355-364 (1997)]. Therefore, in an expression vector constructed using the promoter of cspA gene, regulation of expression is incomplete and, in case of a gene whose product is harmful to the host, there are some cases where it is difficult to incubate to such an state that
E. coli
containing the expression vector can be induced or even the construction of the expression vector is impossible.
For example, in U.S. Pat. No. 5,654,169, there is a description that, even when &bgr;-galactosidase gene which is commonly used for evaluation of promoter is inserted into an expression plasmid using the promoter of cspA gene, it is difficult to keep the constructed product in
E. coli
due to the expressed product.
On the other hand, it has been known that an ability of the promoter of cspA gene to initiate transcription is held at the region which is downstream from −37 from the initiation point of transcription, however, an essential region has not been confirmed yet. Further, the above U.S. Patent shows a region of −40~96 from the initiation point for transcription for the said gene as an essential region for the function as a promoter of cspA gene. However, the said region contains a region of nearly 100 bases which is transcribed to mRNA and, in addition, does not code f or the protein. As such, the minimum region of cspA promoter which is necessary for achieving a transcription having a good efficiency at low temperature has not been clarified yet.
PROBLEMS TO BE SOLVED BY THE INVENTION
Accordingly, an object of the present invention is to offer a vector where a transformant for expression of the said gene can be prepared and where the said gene product can be expressed in a high efficiency even under a low temperature condition even in the case of gene whereby construction of an expression system or efficient production of gene product is difficult in the prior art.
MEANS TO SOLVE THE PROBLEMS
The present inventors have attempted that, in order to achieve the above object, a lac operation sequence is inserted into a downstream of promoter of cspA gene whereby, during the construction of plasmid and incubation until the inducible state, gene expression from

Affiliated with

Also associated with

Rating

Cold-inducible expression vector does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Cold-inducible expression vector, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cold-inducible expression vector will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2933929