title: Overexpression, purification, and characterization of beta-subunit group II chaperonin from hyperthermophilic Aeropyrum pernix K1.
abstract: In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin beta-subunit gene (ApCpnB, 1,665 bp ORF) from the hyperthermophilic archaeon A. pernix K1 was amplified by PCR and subcloned into vector pET21a. The constructed pET21a-ApCpnB (6.9 kb) was transformed into E. coli BL21 Codonplus (DE3). The transformant cell successfully expressed ApCpnB, and the expression of ApCpnB (61.2 kDa) was identified through analysis of the fractions by SDS-PAGE (14% gel). The recombinant ApCpnB was purified to higher than 94% by using heat-shock treatment at 90 degrees C for 20 min and fast protein liquid chromatography on a HiTrap Q column step. The purified ApCpnB showed ATPase activity and its activity was dependent on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at 43 degrees C and 50 degrees C, respectively. Specifically, the activity of malate dehydrogenase (MDH) at 85 degrees C was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of procarboxypeptidase B (pro-CPB) and ApCpnB in E. coli BL21 Codonplus (DE3) had a marked effect on the yield of pro-CPB as a soluble and active form, speculating that ApCpnB facilitates the correct folding of pro-CPB. These results suggest that ApCpnB has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.
authors.affiliation: Department of Biomaterial Control, Dong-Eui University, Busan 614-714, Korea.
authors.forename: Eun-Jung
authors.initials: EJ
authors.lastname: Shin
authors.forename: Jin-Woo
authors.initials: JW
authors.lastname: Lee
authors.forename: Jeong-Hwan
authors.initials: JH
authors.lastname: Kim
authors.forename: Sung-Jong
authors.initials: SJ
authors.lastname: Jeon
authors.forename: Yeon-Hee
authors.initials: YH
authors.lastname: Kim
authors.forename: Soo-Wan
authors.initials: SW
authors.lastname: Nam
mesh_terms: D041522:Aeropyrum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000378:metabolism; D043424:Carboxypeptidase B / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000378:metabolism; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D056385:Group II Chaperonins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D019641:DNA, Archaeal; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase; D008291:Malate Dehydrogenase; D002950:Citrate (si)-Synthase; D043424:Carboxypeptidase B; D056385:Group II Chaperonins