title: Expression, purification and characterization of soluble recombinant peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum.
abstract: Vibrio anguillarum, a causative agent of vibriosis in finfish, crustaceans, and bivalves, is a Gram-negative, motile marine bacterium. Most bacteria have developed survival strategies in various environments. The aim of this study was to investigate the changes in protein expression of V. anguillarum O1 incubated under different conditions using two dimensional electrophoresis and MALDI-TOF MS/MS analysis. Result indicated that peptidyl-prolyl cis/trans isomerase (PPIase) expression was increasingly appeared when incubated at low temperature (15°C) and alkaline conditions (pH 10). Subsequently, the ppi gene from V. anguillarum O1 was isolated and overexpressed in Escherichia coli to characterize the biochemical properties. The cloned ppi gene encoded 206 amino acids containing the conserved regions identified in FK506 binding pocket. To determine the optimal conditions of the purified recombinant PPIase protein (VaFKBP22), we used Succinyl-Ala-Phe-Pro-Phe-p nitroanilide as substrate and the highest enzymatic activity was found at 5°C and pH 6. VaFKBP22 was detected in the cytoplasm and periplasm of V. anguillarum O1. In addition, VaFKBP22 also showed chaperone activity and did not show cytotoxic activity.
authors.affiliation: Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea.
authors.forename: So-Hyun
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea.
authors.forename: Jong Min
authors.initials: JM
authors.lastname: Lee
authors.affiliation: Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea.
authors.forename: Dong Seop
authors.initials: DS
authors.lastname: Kang
authors.affiliation: Biotechnology Research Division, National Fisheries Research & Development Institute, Busan 619-705, Republic of Korea.
authors.forename: Dong-Gyun
authors.initials: DG
authors.lastname: Kim
authors.affiliation: Department of Oral Biochemistry, Dental Science Research Institute, Medical Research Center for Biomineralization Disorders, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea.
authors.forename: Sun-Hee
authors.initials: SH
authors.lastname: Ahn
authors.affiliation: Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea. Electronic address: iskong@pknu.ac.kr.
authors.forename: In-Soo
authors.initials: IS
authors.lastname: Kong
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003080:Cold Temperature; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016559:Tacrolimus / Q000494:pharmacology; D053719:Tandem Mass Spectrometry; D014733:Vibrio / Q000201:enzymology* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus
keywords: FKBP; MIP; Peptidyl-prolyl cis/trans isomerase; Vibrio anguillarum