Engineering of protease variants exhibiting altered substrate specificity.
By using an improved genetic screening system, variants of the HAV 3CP protease which exhibit altered P2 specificity were obtained. We randomly mutated the His145, Lys146, Lys147, and Leu155 residues that constitute the S2 pocket of 3CP and then isolated variants that preferred substrates with Gln over the original Thr at the P2 position using a yeast-based screening method. One of the isolated variants cleaved the Gln-containing peptide substrate more efficiently in vitro, proving the efficiency of our method in isolating engineered proteases with desired substrate selectivity.
Sellamuthu
Saravanan
S
Department of Life Science, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea.
Shin
Bae Hyun
BH
Lee
Eui Seung
ES
Rho
Seong-Hwan
SH
Hwang
Wangtaek
W
Lee
Yong Jae
YJ
Han
Hye-Eun
HE
Kim
Jae Il
JI
Park
Woo Jin
WJ
D000086042:3C Viral Proteases; D019943:Amino Acid Substitution; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D019020:Directed Molecular Evolution / Q000379:methods*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D015003:Yeasts / Q000235:genetics
D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases