Patent ID: 8969062
Filing Date: 2015-03-03
Classification: C07H,C07K,C12N,C12P,C12Y

Abstract:
1. A method of purifying a polypeptide with reduced self-cleavage activity relative to its wild-type polypeptide, where the polypeptide is a mutant of tobacco etch virus (TEV) 27 kDa NIa proteinase, and the polypeptide forms inclusion bodies in a cell, comprising the following steps: a) obtaining cells expressing the polypeptide; b) lysing the cells; c) pelleting inclusion bodies by centrifugation of the lysed cells; d) centrifuging the inclusion bodies; e) solubilizing the inclusion bodies in buffer containing solubilization agent; f) centrifuging the solubilized inclusion bodies to obtain a supernatant containing the polypeptide; g) loading the supernatant over a single denaturing column; h) collecting the polypeptide; and i) renaturing the collected polypeptide, wherein milligram quantities of greater than 95% pure, active polypeptide are collected.