Evaluation of different culture conditions for high-level soluble expression of human cyclin A2 with pET vector in BL21 (DE3) and spectroscopic characterization of its inclusion body structure.
In this paper, we evaluated various parameters of culture condition affecting high-level soluble expression of human cyclin A(2) in Escherichia coli BL21(DE3), and demonstrated that the highest protein yield was obtained using TB(no glycerol)+0.5% glucose medium at 25 degrees C. By single immobilized metal ion affinity chromatography, we got highly purified human cyclin A(2) with a yield ranged from 20 to 30 mg/L. By amyloid-diagnostic dye ThT binding and Fourier transform infrared spectroscopy, we observed a significant decrease in alpha-helix content and an increase in beta-sheet structure in cyclin A(2) inclusion body in comparison to its native protein, and confirmed the resemblance of the internal organization of cyclin A(2) inclusion body and amyloid fibrils.
Wang
Xiaohui
X
Division of Biological Inorganic Chemistry, Key Laboratory of Rare Earth Chemistry and Physics, Changchun Institute of Applied Chemistry, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin 130022, China.
Fu
Manliang
M
Ren
Jinsong
J
Qu
Xiaogang
X
D052160:Benzothiazoles; D002846:Chromatography, Affinity; D003470:Culture Media; D019925:Cyclin A / Q000096:biosynthesis* / Q000302:isolation & purification; D056751:Cyclin A2; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology; D017550:Spectroscopy, Fourier Transform Infrared; D013844:Thiazoles / Q000378:metabolism
D052160:Benzothiazoles; C503721:CCNA2 protein, human; D003470:Culture Media; D019925:Cyclin A; D056751:Cyclin A2; D013844:Thiazoles; C009462:thioflavin T; D012293:Rifampin