Characterization of ubiquitin C-terminal hydrolase 1 (YUH1) from Saccharomyces cerevisiae expressed in recombinant Escherichia coli Hyun-Ah Yu a , Sung-Gun Kim b , Eun-Jeong Kim a , Woo-Jong Lee c , Dae-Ok Kim d , Kyungmoon Park e , Yong-Cheol Park a, * , Jin-Ho Seo a,b, * a Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea b Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea c Korea Biotechnology Commercialization Center, Korean Institute of Industrial Technology, Incheon 406-130, Republic of Korea d Department of Food Science and Technology, Kyung Hee University, Yongin, Gyeonggi 446-701, Republic of Korea e Department of Chemical System Engineering, Hongik University, Jochiwon, Choongnam 339-701, Republic of Korea Received 3 April 2007, and in revised form 11 July 2007 Available online 18 July 2007 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 sub- strate, 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. Ó 2007 Elsevier Inc. All rights reserved. Keywords: YUH1; Ubiquitin C-terminal hydrolase; Saccharomyces cerevisiae; Recombinant Escherichia coli Ubiquitin is widely used as a fusion partner in the pro- duction of foreign proteins and peptides by Escherichia coli and Saccharomyces cerevisiae [1–3]. Additionally, it plays an important role in protein degradation, cell-cycle control, stress response and DNA repair in eukaryotic cells. The compact and globular structure of ubiquitin protects the amino terminus of the fusion protein against proteolytic attack [4]. A peptide library containing 870 amino acids and fused with ubiquitin was produced solubly in recombi- nant E. coli at 10–31% of specific yields based on biomass [3]. An increase in the production yield of yeast metallothi- onein by the fusion of ubiquitin seemed to be ascribed to stability improvement and/or more efficient translation of the fusion proteins [5]. Furthermore, deubiquitinating enzymes cleaved rapidly and precisely between the target fusion proteins and the carboxyl-terminal glycine residue of ubiquitin so that the target proteins with over 90% of purity can be obtained simply [3]. Deubiquitinating enzymes (DUBs) 1 categorized in cys- teine proteases specifically cut ubiquitin conjugates at the 1046-5928/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2007.07.005 * Corresponding authors. Fax: +82 2 873 5260 (Y.-C. Park); fax: +82 2 873 5095 (J.-H. Seo). E-mail addresses: ycpark@snu.ac.kr (Y.-C. Park), jhseo94@snu.ac.kr (J.-H. Seo). 1 Abbreviations used: DUBs, deubiquitinating enzymes; UBP, ubiquitin specific protease; UCH, ubiquitin carboxyl-terminal hydrolase; PCR, polymerase chain reaction; DTT, dithiothreitol; YUH1, yeast ubiquitin C-terminal hydrolase 1. www.elsevier.com/locate/yprep Protein Expression and Purification 56 (2007) 20–26