Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 56, Issue 1, Pages 27-34Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2007.05.011
Keywords
human cyclin A(2); inclusion body; soluble expression; ThT binding; Fourier transform infrared spectroscopy
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In this paper, we evaluated various parameters of culture condition affecting high-level soluble expression of human cyclin A, 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. (c) 2007 Elsevier Inc. All rights reserved.
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