Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1990-03-16
1992-11-03
Weimer, Elizabeth C.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
935 6, 935 10, 935 22, 935 41, 435 691, C12N 1500, C12P 2106
Patent
active
051604892
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention relates to new expression vectors carrying a novel type of promoters which are constructed of the P.sub.2 promoter of a ribosomal RNA operon (rrn B operon) of Escherichia coli and some regulatory sequences from the lac operon of E. coli. The invention further relates to a method for constructing the above mentioned novel promoters and new expression vectors.
BACKGROUND OF THE INVENTION
Genes of any origin carrying any type of information can be introduced into bacterial cells by means of in vitro DNA recombination, as it is widely known. Stable maintenance of the foreign DNA and the expression of the information for which it codes can be carried out by means of appropriate carrier molecules, the expression vectors. When expressing a gene coding for a protein in bacterial cells, the enzymes of the bacterium firstly synthesize an mRNA copy on the DNA template during the course of transcription and a polypeptide chain afterwards, using the information coded in the RNA message, during the course of translation. Transcription is initiated at a DNA region called a promoter. RNA polymerase recognizes this DNA region and binds to it before the initiation of transcription. In vitro DNA recombination methods can be used economically in practice for the production of proteins only if these proteins are produced in relatively high amounts, that is both transcription and translation are effective. Therefore it is very important for promoters to be "strong" (besides other needed features), that is a suitably high level of transcription must be initiated on them.
Different expression vectors have been constructed by the use of many kinds of promoters, e.g. lac, trp, bla, llp, lambda p.sub.L promoters. The promoter of the lac operon has been widely used in practice mainly becouse of its well controlled functioning though it is a relatively week promoter. So called hybrid promoters of different origin have been described e.g. in European Patent Application No. 82302532.5 (publication number 0 06.sup.-- 540). These promoters are characterized by the fact that the so called -35 and -10 regions (which are) the most important regions of a well functioning promoter) are of different origin, that is the two promoter parts of different origin are joined together in the region between the -35 and -10 regions. According to the abovementioned patent application, expression vectors constructed with the use of such hybrid promoters are excellent for industrial purposes. Since their functioning is well controlled and they make efficient transcription possible at the same time.
OBJECT OF THE INVENTION
The object is to develop expression vectors carrying stronger promoters-to render good protein yields-which are at the same time well controlled.
SUMMARY OF THE INVENTION
At the beginning of our research project we carried out experiments using the expression vectors described in: Lukacsovich et al: New regulatory features of the promoters of an Escherichia coli rRNA gene (J. Bact. 169, 272-277) January 1987. The best functioning of these vectors carried promoters with the main features as follows: ribosomal operon (rrnB) of E. coli and a part coming from the regulatory sequences of the lac operon of E. coli, downstream of the -10 region, rich region downstream of the -10 region in rrnB P.sub.2 has been replaced by analogous sequences originating from the lac operon which do not show repressory effect, so it does not affect the strong functioning of the other parts of the promoter.
Though these promoters have been constructed by the combination of different parts of two different promoters, they can not be called hybrid promoters since the most important parts for functioning both the -35 and -10 regions originate from the same promoter, rrnB P.sub.2. We found these promoters to render very good expression. Carrying out more experiments, we surprisingly found that after eliminating the four base pairs: TGCA downstream of the -10 region in vectors carrying the above characterized promoters, the
REFERENCES:
patent: 4551433 (1985-11-01), De Boer
Brosius et al., PNAS 81: 6929. 1984.
Thayer et al., Mol. Gen. Genet 199: 55. 1985.
Luckcsovich et al., J. of Bact. 169(1): 272. 1987.
Baliko Gabriella
Boros Imre
Gaal Tamas
Lukacsovich Tamas
Venetianer Pal
Dubno Herbert
Myers Jonathan
Richter Gedeon Vegyeszeti Gyar R.T.
Weimer Elizabeth C.
Ziska Suzanne
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