T7 expression system
OwnerWayne Barnesinventor
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Abstract
The present invention provides an improved prokaryotic cell expression system employing a tightly controlled host strain construct that controls uninduced, leaky expression of proteins while still auto-inducing well. Various aspects of the present invention address and overcome the problem of uninduced basal expression by providing a host strain that comprises a T7 polymerase gene, and mutants thereof, inserted between lac Z and lac Y of the lac operon (a “ZRY” construct), downstream of an otherwise wild-type lac operon control region.
Claims20
An expression system for producing a target polypeptide in a host cell, the expression system comprising:
- an isolated nucleic acid construct comprising
- (i) a first polynucleotide sequence (R) comprising:
- (a) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:
- 2;
- (b) an isolated polynucleotide comprising SEQ ID NO:
- 1 encoding a T7 RNA polymerase;
- (c) an isolated polynucleotide comprising a nucleotide sequence which has at least 95% identity to that of SEQ ID NO:
- 1 and which encodes a polypeptide having RNA polymerase activity;
- (d) an isolated polynucleotide that hybridizes under stringent conditions to SEQ ID NO:
- 1;
- said stringent conditions comprising incubation at 65° C. in a solution comprising 6×SSC (0.9 M sodium chloride and 0.09 M sodium citrate);
- and which encodes a polypeptide having RNA polymerase activity;
- and
- (e) an isolated polynucleotide complementary to the polynucleotide sequence of (a), (b), (c), or (d);
- (ii) a lac Z polynucleotide sequence (Z) encoding β-galactosidase;
- (iii) a lac Y polynucleotide sequence (Y) encoding a β-galactoside permease;
- and
- (iv) a wild-type lac control region (C) comprising a CAP binding site, a promoter, an operator, and a ribosome binding site;
- wherein (R) is located between (Z) and (Y), and ZRY is downstream of the wild-type lac control region (C);
- the promoter of (C) is a lac-inducible and catabolite-repressible promoter recognized by a host cell polymerase;
- and the polynucleotide sequence of (a), (b), (c), (d), or (e) is under the control of the lac-inducible and catabolite-repressible promoter.
An isolated polypeptide comprising a variant of a polypeptide of SEQ ID NO:
- 2 wherein,
- the variant polypeptide is at least 95% identical to SEQ ID NO:
- 2 over the entire length thereof;
- the variant polypeptide has at least two mutations at an amino acid residue position corresponding to SEQ ID NO:
- 2 selected from the group consisting of:
- (i) amino acid residue 84, Arg to His;
- (ii) amino acid residue 414, Ile to Val;
- (iii) amino acid residue 653, Asp to Gly;
- (iv) amino acid residue 698, Trp to Gly;
- and
- (v) amino acid residue 735, Val to Met;
- the variant polypeptide has RNA polymerase activity;
- the variant polypeptide has reduced rates of uninduced expression in a T7 expression system compared to a T7 RNA polymerase of SEQ ID NO:
- 2;
- and
- the variant polypeptide has about the same or greater rates of induced expression in a T7 expression system compared to a T7 RNA polymerase of SEQ ID NO:
- 2.
Dependencies are inferred from the claim text.