title: Engineering of protease variants exhibiting altered substrate specificity.
abstract: 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.
authors.affiliation: Department of Life Science, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea.
authors.forename: Saravanan
authors.initials: S
authors.lastname: Sellamuthu
authors.forename: Bae Hyun
authors.initials: BH
authors.lastname: Shin
authors.forename: Eui Seung
authors.initials: ES
authors.lastname: Lee
authors.forename: Seong-Hwan
authors.initials: SH
authors.lastname: Rho
authors.forename: Wangtaek
authors.initials: W
authors.lastname: Hwang
authors.forename: Yong Jae
authors.initials: YJ
authors.lastname: Lee
authors.forename: Hye-Eun
authors.initials: HE
authors.lastname: Han
authors.forename: Jae Il
authors.initials: JI
authors.lastname: Kim
authors.forename: Woo Jin
authors.initials: WJ
authors.lastname: Park
mesh_terms: 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
chemical_list: D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases