Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1994-07-28
1997-09-23
Fleisher, Mindy
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, 4353201, 536 231, 536 232, 536 235, 536 236, 536 237, 536 241, C12P 2100, C12N 1564, C12N 1567, C12N 1570
Patent
active
056703333
DESCRIPTION:
BRIEF SUMMARY
This invention relates to methods for expressing polypeptides in E. coli, and to expression vectors and recombinant DNA molecules useful in such methods. The invention further provides a process for producing polypeptides derived from genes native to the genus Thermus.
Bacteria such as E. coli are widely used as host organisms in processes for the production of foreign polypeptides by recombinant DNA technology. Such processes are particularly useful where desired polypeptides are not easily obtained from their natural source organisms.
In the past, techniques for the cloning and expression of foreign genes in micro organisms have involved construction of recombinant expression vectors comprising the coding sequence of the desired foreign gene under the control of the specific expression mechanisms of the organism into which the vector is to be introduced.
An important element of these expression mechanisms is the promoter. This is a region of DNA located in relatively close proximity to the coding sequence of a structural gene. The promoter is involved in the binding of DNA polymerase to initiate transcription. Selection of a suitable promoter is important in order to obtain efficient expression. In processes where a high level of expression of product polypeptide is required, it is usual to select a strong promoter, i.e. one which can sustain a high rate of transcription.
In the context of expression systems based on E. coli known strong promoters include the naturally occurring lac and trp promoters and the synthetic hybrid tac promoter. In general, these promoters have consensus sequences showing only slight variation from the following: ##STR1##
It has been an aim of many researchers to provide additional strong promoters particularly in order to allow expression at high levels in circumstances where use of available promoters is problematical. To this end, promoters such as the tac promoter (a hybrid between the trp and lac promoters) have been constructed.
It is an object of the present invention, according to one aspect thereof to provide a novel expression system for expressing genes (especially, but not exclusively foreign genes) in E. coli.
It has now surprisingly been found that an E. coli gene control sequence which includes a promoter which does not have the consensus sequence shown above can sustain a high rate of transcription.
Thus according to one aspect of the present invention there is provided an expression vector For expressing in E. coli a polypeptide other than E. coli malate dehydrogenase coded for by a DNA coding sequence (particularly a foreign DNA coding sequence), said vector comprising a DNA sequence coding for said polypeptide and including an initiation codon wherein said DNA sequence is operatively linked to a sequence located upstream of the initiation codon and which is capable of controlling expression of said polypeptide, characterised in that said upstream sequence (a) consists of the 285 base pair sequence TGA ACG GTA GGG TAT ATT GTC ACC ACC TGT TGG AAT GTT GCG CTA ATG CAT AAG CGA CTG TTA ATT ACG TAA GTT AGG TTC CTG ATT ACG GCA ATT AAA TGC ATA AAC GCT AAA CTT GCG TGA CTA CAC ATT CTT GAG ATG TGG TCA TTG TAA ACG GCA ATT TTG TGG ATT AAG GTC GCG GCA GCG GAG CAA CAT ATC TTA GTT TAT CAA TAT AAT AAG GAG TTT CAT (SEQ ID NO:3) upstream sequence (a), said related sequence differing from the specific sequence (a) only to such an extent that expression of said polypeptide is not eliminated.
It is preferred that said related sequence (b) has at least a 50%, preferably a 75%, most preferably 95% sequence homology with sequence (a) above.
Where sequence (b) deviates from the precise sequence (a) such deviation may comprise deletions, insertions, and/or substitutions. To maintain high levels of expression of the polypeptide, it is preferred that only a relatively small number of such deletions, insertions and/or substitutions be made, i.e. not more than 10, most preferably not more than 5. Generally only 1 or 2 such deletions, insertions and/or substitutions should be made.
Although
REFERENCES:
D.J. Nicholls et al, "Cloning sequencing and over-expression . . . ", Appl Microbiol Biotechnol (1989), 31, pp. 376-382.
R.F. Vogel et al, "Cloning and sequence of the mdh structural gene . . . " Arch. Microbiol (1987), 149, pp. 36-42.
David J. Nicholls et al, "Cloning and nucleotide sequences of the mdh and sucD-genes . . . ", FEMS Microbiology Letters, (1990), 70, pp. 7-14.
J.D. Hoheisel, "A cassette with seven unique restriction sites, . . . ", Gene (1989), 80, pp. 151-154.
D.M. Marquis, et al, "Use of a portable ribosome-binding site . . . " Gene (1986), 42, pp. 175-183.
Sambrook, J. et al, "Expression of cloned genes in Escherichia coli", Molecular Cloning, A laboratory manual, Second edition, pp. 17.2-17.44.
R.M. Alldread et al, "Overexpression of the Thermus aquaticus B malate . . . " Gene (1992), 114, pp. 139-143.
Yen (1991), J. Bact. 173 (17): 5328-5335.
Alldread Richard M.
Atkinson Tony
Nicholls David J.
Scawen Michael D.
Degen Nancy J.
Fleisher Mindy
Public Health Laboratory Service Board
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