Patent
The invention relates to, in part, improved methods for the production of beta-lactamase using Escherichia coli ( E. coli ) cells. High yield production of beta-lactamase is achieved using methods of the invention.
1. A method for the production of a beta-lactamase polypeptide in Escherichia coli ( E. coli ), comprising:
(a) providing a host E. coli cell transformed with an expression vector comprising a sequence encoding the beta-lactamase polypeptide;
(b) culturing the E. coli cell to induce expression of the beta-lactamase polypeptide in the cytoplasm; and
(c) recovering the beta-lactamase polypeptide from a cytoplasmic soluble fraction prepared from the E. coli cell; and
wherein the method yields more than 10 grams of the beta-lactamase polypeptide per liter of culture,
wherein the beta-lactamase polypeptide is not recovered or purified from a periplasmic fraction, a cell envelope fraction, a cytoplasmic insoluble fraction, an inclusion body, or an extracellular fraction of the E. coli cell.
2. The method of claim 1 , wherein the method yields more than 15 grams of the beta-lactamase polypeptide per liter of culture.
3. The method of claim 1 , wherein the E. coli cell is selected from BL21(DE3) or W3110.
4. The method of claim 1 , wherein expression of the beta-lactamase polypeptide in the cytoplasm is induced by adding isopropylthiogalactoside (IPTG) to the culture.
5. The method of claim 1 , wherein the beta-lactamase polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 1 (P1A).
6. The method of claim 1 , wherein the beta-lactamase polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 10 (P2A).
7. The method of claim 1 , wherein the beta-lactamase polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 5 (P3A).
8. The method of claim 1 , wherein the beta-lactamase polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 7 (P4A).
9. The method of claim 7 , wherein the beta-lactamase polypeptide comprises the amino acid sequence of SEQ ID NO: 5 (P3A).
10. The method of claim 6 , wherein the beta-lactamase polypeptide comprises the amino acid sequence of SEQ ID NO: 10 (P2A).
11. The method of claim 1 , wherein the expression vector comprises palindromic DNA looping.
12. The method of claim 1 , wherein the expression vector is capable of tightly controlled gene expression.
13. The method of claim 1 , wherein the production further comprises purifying said beta-lactamase with a single chromatography column step.
14. A method for the production of a beta-lactamase polypeptide in Escherichia coli ( E. coli ), comprising:
(a) providing a host E. coli cell transformed with an expression vector comprising a sequence encoding the beta-lactamase polypeptide;
(b) culturing the E. coli cell to induce expression of the beta-lactamase polypeptide in the cytoplasm; and
(c) recovering the beta-lactamase polypeptide from a soluble fraction prepared from the E. coli cell;
wherein:
the method yields more than 15 grams of the beta-lactamase polypeptide per liter of culture;
wherein the beta-lactamase polypeptide is not recovered or purified from a periplasmic fraction, a cell envelope fraction, a cytoplasmic insoluble fraction, an inclusion body, or an extracellular fraction of the E. coli cell.