Obtaining Overstable Methionine Aminopeptidase for the Removal of Methionine From Recombinant Proteins

Abstract

Cleavage of the N-terminal initiating methionine (Met1) is a critical coand post-translational modification affecting 50–70% of cellular proteins. During the production of recombinant proteins in the heterologous system of E. coli expression, Met1 cleavage often fails to occur, which leads to heterogeneity of the preparations obtained, changes in their activity and stability. This problem can be solved by treating recombinant proteins in vitro with a specific enzyme, methionine aminopeptidase (MAP). Currently available MAPs exhibit limited specificities and reaction conditions. We cloned a MAP from a hyperthermophilic bacterium, developed a method for enzyme purification, and studied a number of physicochemical properties. The new MAP enzyme is resistant to elevated temperatures. The MAP maintains a stable native state in a pH range from 3 to 11 units. The novel MAP enzyme can be used to remove N-terminal Met1 from recombinant proteins in vitro over a wide pH range and at elevated temperatures.