Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-03-12
2000-11-07
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4353201, 4352523, 43525233, 536 231, 536 241, 536 235, C12N 1500
Patent
active
06143518&
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the production of recombinant proteins in bacteria. It relates, more specifically, to a novel method which allows an elevated production of recombinant proteins in bacteria and which makes use of an expression system which is particularly stable and efficient and which comprises a bacteriophage T7 promoter and a stabilizing region. The present invention also relates to the plasmids which can be employed in this method and to the recombinant proteins which are produced in this way.
Bacteria, in particular E. coli, can be used to produce proteins of varied origin, both from procaryotes and from eucaryotes. In particular, human proteins can be produced in E. coli. In order to do this, the structural gene, or the cDNA of the gene which encodes the protein in question, must be placed downstream of the appropriate expression signals (promoter and ribosome attachment site) which are recognized by the E. coli expression machinery. The most efficient system used in E. coli is that described by Studier (Studier et al., 1990). This system involves using a phage T7 promoter which is specifically recognized by the RNA polymerase of this same bacteriophage (E. coli does not, per se, possess any promoter which is recognized by this particular polymerase). This expression system involves induction, for example with IPTG, of the expression of the gene of the phage T7 RNA polymerase (with the latter being placed under the control of an inducible promoter such as PlacUV5 of E. coli). This induction results in expression of the gene which is placed under the control of the bacteriophage T7 promoter, which is recognized by the polymerase. Since the polymerase only recognizes the promoter which is upstream of the gene to be expressed, the latter gene is, in general, expressed at a very high level (above a few per cent of the total bacterial protein). A very large number of publications now provide instances of the use of this system which was initially described by Studier (Studier et al., 1990).
As indicated above, bacteria, in particular E. coli, are used to produce human proteins of pharmaceutical interest. If the said proteins are then to be used in various therapies, it is advisable for them to be prepared in accordance with the rules of Good Production Practice (GPP). In order to do this, it is essential to have available a production system which is reproducible and which ensures that a product is obtained which is of highly defined quality and very high purity. One of the important features of a method for producing a protein of pharmaceutical interest is the strain which is used to produce the said protein. Thus, it is necessary for this strain to be such as to ensure that the mode of production is reproducible both qualitatively and quantitatively.
At the qualitative level, for example, it is appropriate to produce a protein which is totally homogeneous, that is to say which has a primary sequence which is identical to that of the natural protein. Thus, if a point mutation, which modifies the encoded sequence of the protein to be expressed, were to be produced in a bacterial cell during the course of the fermentation, this would result in the production of a modified protein which was mixed together with the protein having the native sequence. On administration to humans, this modified protein could give rise to an immune reaction which could be very harmful to the treatment or else, in a subsequent treatment, result on this occasion in a severe immune reaction.
At the quantitative level, it is an accepted fact that a method must be reproducible. This constitutes evidence of the quality of the product which is obtained. Thus, in different production batches, the amounts of the protein in question which are synthesized per unit of biomass should be the same, for the same unit of time and using the same culture and growth conditions. A very important feature of this reproducibility of the production of the protein is the presence of the plasmid carrying the expression cassette in the bac
REFERENCES:
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Tabor et al. (1995) Proc. Natl. Acad. Sci USA, 82:1074-1078.
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Tabor et al., A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes, Proc. Natl. Acad. Sci., 82, p. 1074-1078, (1995).
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Cameron Beatrice
Crouzet Joel
Longton Enrique D.
Rhone-Poulenc Rorer SA
Wax Robert A.
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