Engineering of Kex2 variants exhibiting altered substrate specificity.
Engineering of secreted protease variants exhibiting altered substrate specificity is a challenging task because effective screening methods for the desired property are not available yet. In this study, we sought to obtain variants of Kex2, a yeast Golgi protease, which exhibit altered P2 specificity. We first randomly mutated three Asp residues (D176, D210, and D211) that constitute the S2 pocket of Kex2 and then isolated from the resulting library Kex2 variants that preferred substrates with Met (poorly preferred by wild type Kex2) at the P2 position using a yeast-based screening method. The Kex2 variants isolated from this initial screening were further tested against various substrate sequences. Four out of the 16 isolated Kex2 variants showed greater preference for Met than for Lys (preferred by the wild-type Kex2) at the P2 position. We therefore suggest that our method might serve as an efficient tool for engineering and directing the evolution of secreted proteases.
Han
Hye-Eun
HE
National Research Laboratory of Proteolysis and Department of Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea.
Rho
Seong-Hwan
SH
Lee
Yong Jae
YJ
Park
Woo Jin
WJ
D000595:Amino Acid Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D043484:Proprotein Convertases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D040681:Structural Homology, Protein; D013379:Substrate Specificity
D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; C058751:KEX2 protein, S cerevisiae