Recombinant Equine Cytochrome c in Escherichia coli: High-Level Expression, Characterization, and Folding and Assembly Mutants

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• 作者：Jon N. Rumbley ; Linh Hoang ; and S. Walter Englander
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：November 26, 2002
• 年：2002
• 卷：41
• 期：47
• 页码：13894 - 13901
• 全文大小：190K
• 年卷期：v.41,no.47(November 26, 2002)
• ISSN：1520-4995

文摘

To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a systemfor facile mutagenesis and high-level expression is desirable. This work used a generally applicable strategyfor improving yields of heterologously expressed protein in Escherichia coli. Starting with the yeast Cytc plus heme lyase construct of Pollock et al. [Pollock, W. B., Rosell, F. I., Twitchett, M. B., Dumont, M.E., and Mauk, A. G. (1998) Biochemistry 37, 6124-6131], an E. coli-based system was designed thatconsistently produces high yields of recombinant eucaryotic (equine) Cyt c. Systematic changes to theribosome binding site, plasmid sequence, E. coli strain, growth temperature, and growth duration increasedyields from 2 to 3 mg/L to as much as 105 mg/L. Issues related to purification, fidelity of heme insertion,equilibrium stability, and introduction and analysis of mutant forms are described. As an example, variantstailored for folding studies are discussed. These remove known pH-dependent kinetic folding barriers(His26 and His33 and N-terminus), reveal an additional kinetic trap at higher pH due to some undeterminedresidue(s), and show how a new barrier can be placed at different points in the folding pathway in orderto trap and characterize different folding intermediates. In addition, destabilizing glycine mutants in theN-terminal helix are shown to affect the fractional yield of a heme inverted Cyt c isoform.