Characterization of ubiquitin C-terminal hydrolase 1 (YUH1) from Saccharomyces cerevisiae expressed in recombinant Escherichia coli
- Authors
- Yu, Hyun-Ah; Kim, Sung-Gun; Kim, Eun-Jeong; Lee, Woo-Jong; Kim, Dae-Ok; Park, Kyungmoon; Park, Yong-Cheol; Seo, Jin-Ho
- Issue Date
- Nov-2007
- Publisher
- ACADEMIC PRESS INC ELSEVIER SCIENCE
- Keywords
- YUH1; ubiquitin c-terminal hydrolase; Saccharomyces cerevisiae; recombinant Escherichia coli
- Citation
- PROTEIN EXPRESSION AND PURIFICATION, v.56, no.1, pp.20 - 26
- Journal Title
- PROTEIN EXPRESSION AND PURIFICATION
- Volume
- 56
- Number
- 1
- Start Page
- 20
- End Page
- 26
- URI
- https://scholarworks.bwise.kr/hongik/handle/2020.sw.hongik/23523
- DOI
- 10.1016/j.pep.2007.07.005
- ISSN
- 1046-5928
- 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 coh. 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 (1.72 U/mg) and YUH1H6 (1.61 U/mg) were about 18 times higher than 0.092 U/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 1.4 h 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 degrees A of decrease in specific activity for 7 days, corresponding to a 2.7-fold increase compared with that of the free YUH1 incubated in the same solution at 4 degrees C. (c) 2007 Elsevier Inc. All rights reserved.
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