Expression of the kinase domain of mouse protein kinase C in E. coli.
The kinase domain of mouse protein kinase C type alpha has been expressed at high levels in E. coli. The protein has been purified 500-fold taking advantage of the fact that highly expressed fusion proteins precipitate out in the bacterial cell and can be solubilized in 7 M urea. The purified protein can be detected with an antibody generated against a PKC alpha derived synthetic peptide. The purified kinase domain exhibits no measurable kinase activity in a protamin phosphorylation assay. This could be an indication that post-translational modifications of the protein kinase domain which do not happen in bacteria are a requirement for functional enzyme activity or that the regulatory domain of protein kinase C is indispensable for kinase function.
Dietrich
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A
Department of Biochemistry, German Cancer Research Center, Heidelberg.
Rose-John
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S
Marks
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F
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors; D051379:Mice; D011493:Protein Kinase C / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D014169:Transformation, Bacterial
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011493:Protein Kinase C