Expression system
A perfect palindrome operator sequence-based protein expression system is provided. The expression system comprises a promoter; and a perfect palindrome operator sequence, wherein the promoter is not T7. The expression system is preferably employed for the production of recombinant proteins by fermentation.
1. A perfect palindrome operator sequence-based recombinant protein expression system for expression of proteins in prokaryotic cells comprising:
(a) an E. coli polymerase-dependent T7A3 promoter; and
(b) two or more perfect palindrome operator sequences, at least one operator sequence being located downstream of the promoter, and at least one operator sequence being located upstream of the promoter;
wherein:
the operator sequence upstream of the promoter and the operator sequence downstream of the promoter are spaced from 85 to 150 base pairs apart, and
an operator sequence overlaps the transcriptional start point.
2. An expression system according to claim 1 , wherein the operator sequences are spaced 91 or 92 base pairs apart.
3. A plasmid for expression of proteins in prokaryotic cells comprising:
(a) an E. coli polymerase-dependent T7A3 promoter; and
(b) two or more perfect palindrome operator sequences, at least one operator sequence being located downstream of the promoter, and at least one operator sequence being located upstream of the promoter;
wherein the operator sequence upstream of the promoter and the operator sequence downstream of the promoter are spaced from 85 to 150 base pairs apart.
4. A plasmid according to claim 3 , further comprising an expression cassette for a protein.
5. A plasmid according to claim 3 , wherein the operator sequences are spaced 91 or 92 base pairs apart.
6. A host cell transformed by a plasmid of claim 4 .
7. A host cell according to claim 6 , wherein the host cell is E. coli.
8. A method for the production of a recombinant protein in a prokaryotic cell which comprises expressing an expression system comprising
(a) an E. coli polymerase-dependent T7A3 promoter;
(b) two or more perfect palindrome operator sequences, at least one operator sequence being located downstream of the promoter, and at least one operator sequence being located upstream of the promoter; and
(c) an expression cassette for a recombinant protein,
wherein the operator sequence upstream of the promoter and the operator sequence downstream of the promoter are spaced from 85 to 150 base pairs apart, and
an operator sequence overlaps the transcriptional start point.
9. A method according to claim 8 , wherein the operator sequences are spaced 91 or 92 base pairs apart.
10. A method for producing a protein, which comprises:
(a) culturing a host cell transformed with a plasmid of claim 4 ; and
(b) recovering the protein.
11. A method according to claim 10 , wherein the host cell is E. coli.
12. A method according to claim 1 , wherein said two or more perfect palindrome operator sequences are lac or gal operator sequences.
13. A method according to claim 1 , wherein said two or more perfect palindrome operator sequences are lac operator sequences.