Bacterial Expression of a Mitochondrial Cytochrome c. Trimethylation of Lys72 in Yeast iso-1-Cytochrome c and the Alkaline Conformational Transition

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• 作者：W. Brent R. Pollock ; Federico I. Rosell ; Mark B. Twitchett ; Mark E. Dumont ; and A. Grant Mauk
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：April 28, 1998
• 年：1998
• 卷：37
• 期：17
• 页码：6124 - 6131
• 全文大小：86K
• 年卷期：v.37,no.17(April 28, 1998)
• ISSN：1520-4995

文摘

Saccharomyces cerevisiae iso-1-cytochromec has been expressed in Escherichia colibycoexpression of the genes encoding the cytochrome (CYC1) andyeast cytochrome c heme lyase(CYC3).Construction of this expression system involved cloning the twogenes in parallel into the vector pUC18to give the plasmid pBPCYC1(wt)/3. Transcription was directedby two promoters, Lac and Trc, thatwere located upstream from CYC1. Both proteins wereexpressed in the cytoplasm of E. coli cellsharboringthe plasmid. Semianaerobic cultures grown in a fermentor produced15 mg of recombinant iso-1-cytochrome c per liter of culture. Attempts to increaseproduction by addition of IPTG suppressed thenumber of copies of the CYC1 gene within the population.Wild-type iso-1-cytochrome c expressedwithpBPCYC1(wt)/3 in E. coli was compared to the sameprotein expressed in yeast. At neutral pH, the twoproteins exhibit indistinguishable spectroscopic and physical(T_m, E_m')characteristics. However,electrospray mass spectrometry revealed that the lysyl residue atposition 72 is not trimethylated by E.coli as it is by S. cerevisiae. Interestingly, thepK_a of the alkaline transition of the proteinexpressed inE. coli is ~0.6 pK_a unit lower thanthat observed for the cytochrome expressed in yeast (8.5-8.7).¹HNMR spectroscopy of the bacterially expressed cytochrome collected athigh pH revealed the presence ofa third alkaline conformer that is not observed in the correspondingspectrum of the cytochrome expressedin yeast. These observations suggest that Lys72 can serve as anaxial ligand to the heme iron of alkalineiso-1-ferricytochrome c if it is not modifiedposttranscriptionally to trimethyllysine.