Gene cloning and characterization of thermostable peptidyl prolyl cis-trans isomerase (PPIase) from Bacillus stearothermophilus SIC1 |
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| Institution: | 1. School of Public Health, Qingdao University, Qingdao, China;2. School of Public Health, Sun Yat-sen University, Guangzhou, China;1. School of Metallurgy and Environment, Central South University, Changsha 410083, China;2. Chinese National Engineering Research Center for Control and Treatment of Heavy Metal Pollution, Changsha 410083, China;1. Department of Pharmaceutics, School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai 201203, China;2. Center for Advanced Low-dimension Materials, State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201600, China;3. Department of Neurosurgery, Affiliated Hospital of Nantong University, Nantong 226001, China |
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| Abstract: | The peptidyl prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) gene (ppiT) from Bacillus stearothermophilus SIC1 was cloned on the basis of a partial amino acid sequence of the purified enzyme. ppiT was found as an open reading frame (501 bases) which coded for a protein consisting of 167 amino acid residues (molecular weight, 18,349) (GenBank accession number D42050). The cloned ppiT was overexpressed in Escherichia coli cells using pET-8c as an expression vector. The enzyme was purified by heat treatment and column chromatography on DEAE-Sepharose CL-6B. Purification was about 148-fold and the molecular weight of the enzyme was estimated to be about 18.0 kDa by SDS-PAGE. PPIase activity was determined using synthetic peptide as a substrate in a 2-step reaction coupled with chymotrypsin treatment. The enzyme was stable at pH 7.5–8.0. No heat denaturation was observed when the enzyme was treated at 60°C for 30 min. The PPIase purified from recombinant E. coli has almost the same characteristics as that from B. stearothermophilus SIC1. In refolding solution, the PPIase enhanced the isomerization rate of unfolded RNase T1. |
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