title: Characterization of ubiquitin C-terminal hydrolase 1 (YUH1) from Saccharomyces cerevisiae expressed in recombinant Escherichia coli.
abstract: The YUH1 gene coding for ubiquitin C-terminal hydrolase 1, a deubiquitinating enzyme, was cloned from the Saccharomyces cerevisiae genomic DNA and expressed in Escherichia coli. YUH1 was fused with the 6 histidine tag at the N-terminus (H6YUH1) or C-terminus (YUH1H6) and purified by an immobilized metal affinity chromatography with high purity. By using a fluorogenic substrate, Z-Arg-Leu-Arg-Gly-Gly-AMC, the deubiquitinating activities for H6YUH1 (1.72U/mg) and YUH1H6 (1.61U/mg) were about 18 times higher than 0.092U/mg for H6UBP1, ubiquitin specific protease 1 of S. cerevisiae containing the 6 histidine residue at the N-terminus which is normally used in protein engineering. YUH1 had the optimal temperature of 27 degrees C and acidity of pH 8.5. Analysis of thermal deactivation kinetics of H6YUH1 estimated 3.2 and 1.4h of half lives at 4 and 52 degrees C, respectively. Immobilization onto the Ni-NTA affinity resin and environmental modulation were carried out to improve the stability of YUH1. Incubation of the immobilized YUH1 in 50% glycerol solution at -20 degrees C resulted in 52% of decrease in specific activity for 7days, corresponding to a 2.7-fold increase compared with that of the free YUH1 incubated in the same solution at 4 degrees C.
authors: Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea.
authors: Hyun-Ah
authors: HA
authors: Yu
authors: Sung-Gun
authors: SG
authors: Kim
authors: Eun-Jeong
authors: EJ
authors: Kim
authors: Woo-Jong
authors: WJ
authors: Lee
authors: Dae-Ok
authors: DO
authors: Kim
authors: Kyungmoon
authors: K
authors: Park
authors: Yong-Cheol
authors: YC
authors: Park
authors: Jin-Ho
authors: JH
authors: Seo
mesh_terms: D003001:Cloning, Molecular; D010450:Endopeptidases / Q000096:biosynthesis* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase