Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1989-01-23
2001-02-20
Schwartzman, Robert A. (Department: 1635)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S069800, C435S071200, C536S023100, C536S024100
Reexamination Certificate
active
06190906
ABSTRACT:
DESCRIPTION
The present invention is concerned with an expression vector for the regulatable expression of foreign genes in procaryotes.
Expression vectors are needed for the expression of proteins in procaryotes. Apart from the gene of the protein to be expressed, such vectors must also contain regulation sequences which make possible the transcription and translation processes in the cells used for the protein production. Insofar as expression is to be carried out in procaryotes, such regulation sequences originate from procaryotes and contain, for example, sequences of promoters, operators and ribosomal binding points. Usually, such regulation sequences are present in a vector, for example the plasmid pBR 322 or a derivative thereof, in front the gene. Besides the previously described regulation sequences, these vectors also contain a replication origin and a marker for the selection of the plasmid, for example tetracycline resistance, ampicillin resistance, kanamycin resistance and the like.
The DNA unit, which recognizes RNA polymerase dependent upon the DNA and is transferred into the messenger RNA, is referred to as a transcription unit. Such a transcription unit consists of recognition sequences for the DNA-dependent RNA polymerase, recognition sequences for the initiation of the ribosomal function (Shine-Dalgarno sequence), the start codon ATG, a stop codon and termination sequences at which the transcription is terminated. Certain genes, namely those the gene products of which do not remain localized in the cytoplasm but rather are secreted into the periplasm or into the outer membrane, can, in addition, contain yet other recognition sequences. Such a recognition sequence for the excretion of proteins is called a signal sequence. A signal sequence contains characteristically charged segments, hydrophobic regions and hydrophilic regions and a signal sequence cleavage site (see the review article, Mechanism of Protein Localisation, Microbiol. Reviews, 47, 314-344/1983).
Consequently, the following requirements are demanded of an expression vector:
a) it must contain a strong promotor,
b) fusions with foreign genes are to be easy to carry out; and
c) the promotor, including an attached foreign gene section, should be suitable to make possible a localisation of the protein not only in the cyto-plasm but also in the periplasm or in the medium.
It is known that the extent of the expression of the foreign gene depends substantially upon the promotor. In order to assess whether a particular promotor is especially suitable for the expression of foreign genes, it does not depend, however, solely upon the amount of the foreign protein produced per cell but very often it is desirable that the gene product is not synthesized during the whole of the growth phase but only over a particular period of time and preferably in the late growth phase. This is particularly desirable in the case of the expression of gene products which, when they are present in large amounts, are either toxic to the cells or inhibit the growth of the cells. Therefore, it is often necessary to suppress the activity of a promotor at the beginning of the fermentation phase so that initially an extensive biomass is produced. Subsequently, by suitable means, the promotor should be stimulated and the expression of the foreign gene can take place.
Promotors known for this purpose include, for example, the lac promotor, the trp promotor and the &lgr;-P
L
-promotor. However, these promotors are not very suitable for a large-scale production of heterologous proteins. It is known that the lac promotor is inactivated by glucose but not completely enough to prevent the synthesis of “toxic” proteins. The trp promotor is also not an especially suitable promotor for large-scale production. Quite apart from the fact that the use of high tryptophane concentrations for the repression makes the fermentation considerably more expensive, it has been found that tryptophane does not make possible a complete repression so that here, too, disturbing expression can take place during the initial phase of the fermentation. The &lgr;-P
L
-promotor is also not suitable for a large-scale fermentation. The repression mechanism here takes place by the binding of a thermolabile repressor to an operator present behind the promotor at 32° C. The repressor is inactivated by increasing the temperature to 42° C., the transcription thereby being made possible. For large-scale production, which involves the use of fermentation volumes of 50 to 100 m
3
, such an increase of the temperature involves great difficulties. Furthermore, it has been found that the induction of the &lgr;-P
L
-promotor must take place in an early growth phase so that the biomass necessary for a biotechnical large-scale production cannot be achieved.
Therefore, it is an object of the present invention to overcome the above-described difficulties and to provide an expression vector for the regulatable expression of foreign genes in prokaryotes.
Thus, according to the present invention, there is provided an expression vector which consists of a DNA vector which, as regulation sequence, contains the promotor/operator region and the initiation point of the translation of the mg1 operon. The terms “expression vector” or “vector,” as used herein, refer to recipient DNA molecules which contain an origin of replication and a marker so as to show its presence in a host cell. A foreign gene may be introduced into the vector, if desired.
When the expression vector contains at least one foreign gene which is under the expression control of the mg1 operon regulation sequences, its expression can be positively or negatively regulated via the mg1 promotor. However, the foreign gene can also be introduced later.
The mg1 promotor contained in the expression vector according to the present invention is a part of the mg1 operon which, besides the promotor, also contains 4 structural genes, mg1A, mg1B, mg1E and mg1C, which are subject to control by the mg1 promotor. The mg1B gene hereby codes for a galactosidase binding protein of 33,000 Dalton and mg1A, mg1C and mg1E each code for membrane-bound proteins. Thus, proteins are expressed by the mg1 operon which are responsible for the transport of galactose from the medium into the bacterial cell.
For the production of the vector according to the present invention, the mg1 operon can, in principle, be isolated from the genome of a cell which is able to utilise galactose provided from the outside, for example from
Salmonella typhimurium,
DSM 554, or from
Escherichia coli,
MC 4100 (DSM 4090) (J. Biol. Chem., 258, 10853-10855/1983; J. Bacteriol., 153, 408-415/1983).
The isolation of the mg1 operon from
Salmonella typhimurium
can, according to the present invention, take place in such a manner that the genomic DNA is cleaved with EcoRI and a 6.3 kb sized fragment is isolated which is cloned in the usual way and can subsequently be expressed (cf. in this regard, J. Bacteriol., 163, 37-85/1985, as well as the literature cited therein). From the plasmid pNM 506 cited in this literature reference, there can be split out, by cleavage with EcoRI and BamHI, an approximately 900 bp-sized fragment which is then isolated. From the DNA sequence of this fragment was determined the sequence beginning immediately after the EcoRI recognition sequence (see
FIG. 1
of the accompanying drawings). At position
705
to
707
is present the start codon ATG. Five nucleotides before, there lies the nucleotide sequence GGAG recognisable as Shine-Dalgarno sequence. This fragment contains the mg1 promotor which, however, only represents a part of this DNA sequence.
In contradistinction to previously known regulation systems, the mg1 promotor can be almost completely repressed with glucose and other catabolyte-repressing sugars, for example fructose or glucose-6-phosphate. Thus, with the use of the mg1 promotor according to the present invention, it is possible to control gene expression in an especially simple way. For example, in case of the fermentation, glucose as a
Boos Winfried
Jarsch Michael
Schumacher Gunther
Fulbright & Jaworski L.L.P.
Roche Diagnostics GmbH
Schwartzman Robert A.
Wang Andrew
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