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
1999-03-19
2003-07-22
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S196000, C435S325000, C435S369000
Reexamination Certificate
active
06596508
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to inducible production of recombinant protein in eucaryotic cells. More specifically, it concerns an expression system that has a low-basal level and a high-induced level. The invention also relates to a method of identifying ligands (agonists or antagonists) which binds to endogenous or overexpressed G protein-coupled receptors. In particular, the method relates to ligands which binds to Gs- Gq- or Gi-coupled receptors. The invention also relates to a method of identifying phosphodiesterase inhibitors or PKA activators. Finally, the present invention relates to a plasmid having the reporter gene encoding for the human placental secreted alkaline phosphatase (SEAP) which expression is under the control of a cyclic-AMP (cAMP) inducible promoter.
(b) Description of Prior Art
A problem in recombinant protein production is the toxicity of some foreign proteins to the recombinant host. Foreign proteins overexpression are inherently aberrant in the host and therefore are often unhealthy for the cells at high concentration levels, even if they are not toxins in the conventional sense. Consequently, stable clones overexpressing recombinant proteins are often difficult to obtain or show poor growth characteristics. In the last several years, inducible promoter systems have been employed to overcome these problems. However, there are several problems with the commonly used inducible promoter systems. For example, certain inducible systems will have high-basal transcriptional activity which results in recombinant protein production even though the cells have not been induced. Another problem is that inducible system with low-basal level often tend to have a low-induced level.
The second messenger cyclic adenosine monophosphate (cAMP) can induce transcription by activating transcription factors acting through cAMP-responsive elements (CREs) found in various gene promoters. In addition to cAMP, CRE can be activated by other signalling pathways still to be fully characterized (Montminy, M.,
Annu. Rev. Biochem.,
1997, 66: 807-822). Promoters containing one or multiple CREs can thus be used to control the expression of a recombinant protein or, when driving the expression of a reporter gene such as the secreted placental alkaline phosphatase (SEAP), to monitor G protein-coupled receptors activation since many of them modulate intracellular cAMP levels.
In this application, the cAMP inducible promoter consist of multiple CREs upstream of a fragment of the vasointestinal peptide (VIP) promoter containing one endogenous CRE. This fusion promoter shows little basal transcriptional activity in its uninduced state. When induced, this fusion promoter drives the expression of the human placental secreted alkaline phosphatase (in the case of the cell-based assay application) or any other cDNA coding for a protein to be expressed. The CRE sequence used in this study derive from the vasointestinal peptide (VIP) promoter and have been described in detail by Tsukada et al (
J. Biol. Chem.,
1987, 262:8743) and Fink et al (
Proc. Natl. Acad. Sci. USA,
1988, 85:6662). In this promoter, the CRE consensus site is constituted by two 5 bp palindromic sequences CGTCA separated by five nucleotides, TACTG (Tsukada, T. et al,
DNA,
1985, 4:293). To a 239 bp fragment (−94 to +145) of this VIP promoter (Fink et al,
PNAS,
1988, 85:6662) containing one endogenous CRE (CGTCATACTGTGACG; SEQ ID NO:1), a synthetic DNA fragment containing four CREs (5′-CGTCACAGTATGACG-3′; SEQ ID NO:2) was created and ligated to its 5′ end (Chen, W. et al,
Anal. Biochem.,
1995, 226:349). This resulted in a promoter (4CRE/VIP) containing a total of 5 CREs. The 4CRE/VIP promoter construct was removed from the pCRE/á-Gal vector (kindly provided by Dr Roger D. Cone,
Anal. Biochem.,
1995, 226:349) and used such as, or following specific modifications.
It would therefore be desirable to be provided with an expression system that has an high-inducible level and a low-basal level.
It would also be desirable to be provided with an expression system that can be used to produce proteins in large quantities. It would also be desirable to be provided with an expression system that is useful for producing toxic proteins.
SUMMARY OF THE INVENTION
It is an aim of the present invention to provide an expression system that a has low-basal level and an high-induced level.
It is another aim of the present invention to provide an expression system that allows the production of a recombinant protein in large quantities.
It is another aim of the present invention to provide an expression system that allows the production of recombinant toxic proteins.
In accordance with the present invention, there is provided a DNA construct for the expression of a desired gene which comprises a cyclic AMP sensitive promoter operably linked to a DNA sequence comprising the coding region of the desired gene.
In another aspect, the invention is directed to host expression vectors which contain the foregoing control elements but, rather than the desired gene, contain a polylinker sequence containing restriction sites to permit the insertion of the DNA encoding any other desired protein in reading frame.
In another aspect, the invention is directed to cell transfected with the expression vector and to methods to produce desired proteins by culturing the transfected cells under conditions wherein transcriptional and translation expression are induced.
In accordance with the present invention, there is provided a DNA construct for the expression of a desired gene, which construct comprises a cyclic AMP sensitive promoter operably linked to a DNA sequence comprising the coding region of the desired gene, having a low basal level and a high induced level.
In accordance with a preferred embodiment of the present invention, the cyclic AMP inducible promoter is CRE. More preferably, the cyclic AMP inducible promoter comprises between 1 and 9 CREs.
More preferably, the construct of the present invention includes a cDNA which is encoding for secreted human placental alkaline phosphatase (SEAP).
In accordance with another embodiment of the present invention, there is provided a mammalian expression vector for inducible expression of a protein of interest, which comprises a synthetic promoter containing between 1 and 9 CREs operably linked to a cDNA encoding for a protein of interest, wherein said promoter allows for tight regulation of expression.
In accordance with another embodiment of the present invention, there is provided a recombinant host vertebrate cell transfected with a construct as described herein. The preferred cell used is a mammalian cell.
In accordance with another embodiment of the present invention, there is provided a method to express a gene encoding a desired protein which method comprises culturing the cells described herein under conditions wherein said coding sequence is expressed to produce a desired protein, and recovering the desired protein from the culture.
The method further includes inducing the transcriptional promoter.
A preferred cell line used in accordance with the present invention is a mammal cell line, or a human cell line, most preferably a human embryonic kidney cell line.
A preferred serum-free human embryonic kidney cell line used in accordance with the present invention, which allows for the expression of recombinant adenoviral vectors, referred to as 293SF-3F6, has been deposited at the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, Va. 20110-2209 USA) on Sep. 25, 1998 under deposit number ATCC CRL-12585. This deposit is available to be public upon the grant of a patent to the assignee, National Research Council Canada, disclosing same. The deposit is also available as required by Foreign Patent laws in countries wherein counterpart applications are filed.
Prior to setting forth this invention, it may be helpful to first define certain terms that will be used herein.
By the term “low basal level” is me
Cawthorn Christian
National Research Council of Canada
Prouty Rebecca E.
Renault Ogilvy
Steadman David J.
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