Modified bacterial protein expression system
AssigneeThe Trustees of Columbia University in the City of New York
InventorMaxwell Gottesman
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Abstract
The present disclosure provides host cells for reliable, high yield recombinant protein production, including unstable proteins. The present host cell (e.g., a bacterial cell) is deficient in at least one protease (or a subunit of a protease) such as Clp or ClpP. The host cell may also contain an expression vector that encodes a protein or polypeptide for overexpression.
Claims25
An engineered bacterium comprising at least one deficient protease, wherein the protease is Clp, ClpP, ClpQ (HslV), ClpAP, ClpXP, ClpAXP, ClpYQ, ClpA, ClpX, ClpY (HslU), or combinations thereof, and wherein the bacterium overexpresses a polypeptide at a level which is at least 2 fold of the level of the polypeptide produced by a bacterium not engineered with the deficient protease.
An engineered bacterium comprising a total or partial deletion of at least one gene encoding a protease, wherein the protease is Clp, ClpP, ClpQ (HslV), ClpAP, ClpXP, ClpAXP, ClpYQ, ClpA, ClpX, ClpY (HslU), or combinations thereof.
A method for expressing a polypeptide in bacteria, the method comprising the steps of:
- (a) engineering a bacterial cell to decrease level and/or activity of at least one protease, wherein the protease is Clp, ClpP, ClpQ (HslV), ClpAP, ClpXP, ClpAXP, ClpYQ, ClpA, ClpX, ClpY (HslU), or combinations thereof;
- (b) transforming the engineered bacterial cell with a nucleic acid sequence encoding the polypeptide;
- (c) culturing the bacterium to produce the polypeptide;
- and
- (d) isolating the polypeptide.
Dependencies are inferred from the claim text.