site > International journal > (2007) Characterization of ubiquitin C-terminal hydrolase 1 (YUH1) from Saccharomyces cerevisiae expressed in recombinant Escherichia coli.

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작성일 : 12-07-17 17:45

(2007) Characterization of ubiquitin C-terminal hydrolase 1 (YUH1) from Saccharomyces cerevisiae expressed in recombinant Escherichia coli.

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http://www.sciencedirect.com/science/article/pii/S1046592807001738 [1237]

Characterization of ubiquitin C-terminal hydrolase 1 (YUH1) from Saccharomyces cerevisiae expressed in recombinant Escherichia coli.

Hyun-Ah Yu, Sung-Gun Kim, Eun-Jeong Kim, Woo-Jong Lee, Dae-Ok Kim, Kyungmoon Park, Yong-Cheol Park* and Jin-Ho Seo*
Protein Expression and Purification 56(1): 20-26. 2007.11.01.
(SCI I/F: 1.587)

The YUH1 gene coding for ubiquitinC-terminalhydrolase1, a deubiquitinating enzyme, was cloned from the Saccharomycescerevisiae genomic DNA and expressed in Escherichiacoli. 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 °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 °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 °C resulted in 52% 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 °C.

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