Method for engineering proteases and protein kinases

摘要

Provided are methods for protein engineering, such as engineering proteases or kinases. The methods may utilize yeast display and/or ER sequestration of proteins or substrates. In some aspects, TEV proteases with altered substrate specificity, potency, and/or efficiency are provided.

主权项

1. A nucleic acid vector for engineering protease variants, wherein the nucleic acid vector encodes two separately expressed proteins, wherein the first protein is a first fusion protein comprising, in an N- to C-terminal direction:
(i) a first endoplasmic reticulum (ER) targeting sequence; (ii) a surface expression sequence; (iii) a first peptide sequence that is a counterselection substrate sequence for the enzyme of the second protein; (iv) a first epitope tag sequence; (v) a second peptide sequence that is a selection substrate sequence for the enzyme of the second fusion protein; (vi) a second epitope tag sequence; and (vii) a first endoplasmic reticulum (ER) retention sequence; wherein the second protein is a protease or has protease activity.