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 comprises an amino acid sequence having at least 95% identity with a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 10,
wherein the host E. coli cell is selected from BL21 (DE3) or W3110, and
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 expression of the beta-lactamase polypeptide in the cytoplasm is induced by adding isopropylthiogalactoside (IPTG) to the culture.
4. The method of claim 1 , wherein the beta-lactamase polypeptide comprises the amino acid sequence of SEQ ID NO: 5.
5. The method of claim 1 , wherein the beta-lactamase polypeptide comprises the amino acid sequence of SEQ ID NO: 10 (P2A).
6. The method of claim 1 , wherein the expression vector comprises palindromic DNA looping.
7. The method of claim 1 , wherein the expression vector is capable of tightly controlled gene expression.
8. 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 beta-lactamase polypeptide comprises an amino sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 10 and the method yields more than 15 grams of the beta-lactamase polypeptide per liter of culture,
wherein the host E. coli cell is selected from BL21 (DE3) or W3110, and
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.