Carbapenemases for use with antibiotics for the protection of the intestinal microbiome
This invention relates, in part, to various compositions and methods for protecting the gastrointestinal microbiome from antibiotic disruption.
1. A method for producing a broad spectrum carbapenemase in Escherichia coli ( E. coli ), comprising:
(a) providing a host E. coli cell transformed with a vector comprising a nucleic acid sequence encoding the carbapenemase operably linked to an inducible promoter;
(b) culturing the E. coli cell in a culture medium comprising zinc and inducing the expression of the carbapenemase; and
(c) recovering the carbapenemase from a soluble fraction prepared from the cytoplasm or periplasmic space of the E. coli cell, wherein:
the encoded carbapenemase comprises at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 68 (P2A).
2. The method of claim 1 , wherein the broad spectrum carbapenemase has the amino acid sequence of SEQ ID NO: 68 (P2A).
3. The method of claim 1 , wherein the method yields at least 10 grams of active carbapenemase per liter of culture.
4. The method of claim 1 , wherein the method yields at least 15 grams of active carbapenemase per liter of culture.
5. The method of claim 1 , wherein the recovered carbapenemase is soluble.
6. The method of claim 1 , wherein the culturing is in a bioreactor or a shake flask.
7. The method of claim 1 , wherein the E. coli cell is induced to express the carbpenemase using isopropyl β-D-1-thiogalactopyranoside (IPTG).
8. A method for producing a broad spectrum carbapenemase in Escherichia coli ( E. coli ), comprising:
(a) providing a host E. coli cell transformed with a vector comprising a nucleic acid sequence encoding the carbapenemase operably linked to an inducible promoter;
(b) culturing the E. coli cell in a culture medium comprising zinc and inducing the expression of the carbapenemase; and
(c) recovering the carbapenemase from a soluble fraction prepared from the cytoplasm or periplasmic space of the E. coli cell, wherein:
the encoded carbapenemase comprises at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 53 (NDM-1).
9. The method of claim 8 , wherein the broad spectrum carbapenemase has the amino acid sequence of SEQ ID NO: 53 (NDM-1).
10. The method of claim 8 , wherein the method yields at least 10 grams of active carbapenemase per liter of culture.
11. The method of claim 8 , wherein the method yields at least 15 grams of active carbapenemase per liter of culture.
12. The method of claim 8 , wherein the recovered carbapenemase is soluble.
13. The method of claim 8 , wherein the culturing is in a bioreactor or a shake flask.
14. The method of claim 8 , wherein the E. coli cell is induced to express the carbapenemase using isopropyl β-D-1-thiogalactopyranoside (IPTG).