Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries.

ABSTRACT: Myriad new applications of proteases would be enabled by an ability to fine-tune substrate specificity and activity. Herein we present a general strategy for engineering protease selectivity and activity by capitalizing on sequestration of the protease to be engineered within the yeast endoplasmic reticulum (ER). A substrate fusion protein composed of yeast adhesion receptor subunit Aga2, selection and counterselection substrate sequences, multiple intervening epitope tag sequences, and a C-terminal ER retention sequence is coexpressed with a protease library. Cleavage of the substrate fusion protein by the protease eliminates the ER retention sequence, facilitating transport to the yeast surface. Yeast cells that display Aga2 fusions in which only the selection substrate is cleaved are is

SUBMITTER: Yi L 

PROVIDER: S-EPMC3645551 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature