Expression of recombinant cytochromes c from various species in Saccharomyces cerevisiae: post-translational modifications.
A complete protocol for the expression of recombinant cytochrome c genes from yeast, Drosophila melanogaster, and rat in a yeast strain, GM-3C-2, which does not express its own cytochromes c is described. The construction of the expression vectors, transformation and large-scale growth of the yeast, and preparation and purification of the recombinant cytochromes c are described. It was found that, contrary to the way yeast modifies its own cytochromes c, the recombinant proteins were partially acetylated at their N-terminus, except for the drosophila protein, which remained entirely unblocked. Furthermore, the yeast and rat proteins were close to fully trimethylated at lysine 72, while the drosophila protein could be separated chromatographically into forms containing tri-, di-, mono-, and unmethylated lysine 72 showing corresponding resonances in the NMR spectrum. These observations emphasize that, in employing expression procedures to obtain native or mutant forms of cytochrome c, it is essential to identify the variety and extent of post-translational modifications and to separate the preparation into pure monomolecular species. Otherwise, it may become impossible to distinguish between the influence of a site-directed mutation and unexamined post-translational modifications.
Koshy
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TI
Department of Biological Sciences, University of Illinois, Chicago 60680.
Luntz
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TL
Garber
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EA
Margoliash
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E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004331:Drosophila melanogaster / Q000737:chemistry / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D051381:Rats / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*
D003574:Cytochrome c Group; D011994:Recombinant Proteins