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Title
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Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states.
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Authors
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F.W.Herberg,
S.M.Bell,
S.S.Taylor.
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Ref.
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Protein Eng, 1993,
6,
771-777.
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PubMed id
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Abstract
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The catalytic subunit of mouse cAMP-dependent protein kinase expressed in
Escherichia coli was separated into three distinct species using Mono-S ion
exchange chromatography. These isoenzymes corresponded to three isoelectric
variants with pIs of 6.4 (30%), 7.2 (60%) and 8.2 (10%). The Stokes' radius of
each form was 27.7, 27.1 and 26.3 A respectively. Using electrospray mass
spectroscopy the differences between the isozymes were shown to be due to
phosphorylation, with each form differing by 80 mass units corresponding to a
single phosphate. The fully phosphorylated recombinant enzyme contained four
phosphates while the dominant isozyme contained only three. Since the enzyme is
not phosphorylated when active site mutations are introduced into the C-subunit,
these phosphates are incorporated in an autocatalytic mechanism and are not due
to E. coli protein kinases. When the recombinant enzyme was compared with the
mammalian porcine heart enzyme significant differences in post-translational
modifications were observed. The mammalian enzyme could also be separated into
two isozymes. However, in contrast to the recombinant enzyme, the mammalian
isozymes displayed an identical mass of 40 840. This correlated with two
different post-translational modifications: two phosphates and an N-terminal
myristyl moiety. The importance of post-translational modifications, and in
particular the phosphorylation state, for the expression of eukaryotic proteins
in E. coli is discussed.
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