Overexpression, Purification, and Characterization of β-Subunit of Group Ⅱ Chaperonin from Hyperthermophilic Aeropyrum pernix K1

• Published in: Journal of microbiology and biotechnology Vol. 20; no. 3; pp. 542 - 549
• Main Authors: Shin, E.J., Dong-Eui University, Busan, Republic of Korea, Lee, J.W., Dong-Eui University, Busan, Republic of Korea, Kim, J.H., Dong-Eui University, Busan, Republic of Korea, Jeon, S.J., Dong-Eui University, Busan, Republic of Korea, Kim, Y.H., Dong-Eui University, Busan, Republic of Korea, Nam, S.W., Dong-Eui University, Busan, Republic of Korea
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.03.2010; 한국미생물·생명공학회
• Subjects: Aeropyrum - chemistry; Aeropyrum - genetics; Aeropyrum - metabolism; Aeropyrum pernix; ALCOHOL DEHYDROGENASE; Alcohol Dehydrogenase - metabolism; ALCOHOL DESHIDROGENASA; ALCOOL DESHYDROGENASE; ATPase activity; Biological and medical sciences; Biotechnology; Carboxypeptidase B - biosynthesis; Carboxypeptidase B - genetics; Carboxypeptidase B - metabolism; Chaperonin; Citrate (si)-Synthase - metabolism; citrate synthase; DNA, Archaeal - chemistry; DNA, Archaeal - genetics; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Group II Chaperonins - biosynthesis; Group II Chaperonins - genetics; Group II Chaperonins - isolation & purification; Group II Chaperonins - metabolism; MALATE DEHYDROGENASE; Malate Dehydrogenase - metabolism; MALATE DESHYDROGENASE; MALATO DESHIDROGENASA; Plasmids - genetics; Polymerase Chain Reaction; pro-carboxypeptidase B; Protein Folding; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; 생물학; Chaperonin; Purification; Aeropyrum pernix; Enzyme; Adenosinetriphosphatase; Citrate; Gene overexpression; Malate dehydrogenase; Alcohol dehydrogenase; Synthase; Subunit; Characterization; citrate synthase; Peptidases; Carboxypeptidase B; ATPase activity; Hyperthermophily; Hydrolases; pro-carboxypeptidase B; Oxidoreductases; Metallocarboxypeptidases
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.0909.09025

In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin β-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℃ 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℃ and 50℃, respectively. Specifically, the activity of malate dehydrogenase (MDH) at 85℃ 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.