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A cDNA clone for the catalytic subunit of murine cAMP-dependent protein kinase
was placed into two expression vectors, pLWS-3 and pLSW-4. For pLWS-3, the
entire coding region of the catalytic subunit was inserted into the NdeI site of
pT7-7 under the control of the T7 promoter. pLWS-4 contains a polycistronic
transcript under control of the lac UV5 promoter encoding for the type I
regulatory subunit followed by the catalytic subunit. Significant expression was
achieved with pLWS-4 in Escherichia coli 222 and JM101; however, the catalytic
subunit was produced in an insoluble form. In the case of the catalytic subunit
produced in E. coli BL21(DE3) by pLWS-3, the catalytic subunit accounted for
approximately 30% of the total bacterial protein. Up to 5 mg of this catalytic
subunit per liter of culture was in the soluble extract. Solubility was improved
substantially when induction was carried out at 30 degrees C instead of 37
degrees C. This recombinant catalytic subunit was purified by phosphocellulose
chromatography, followed by ammonium sulfate precipitation and gel filtration. A
Mr of 38,000 was estimated based on size exclusion chromatography and on
polyacrylamide gel electrophoresis. The recombinant protein had a free
alpha-amino-terminal Gly in contrast to the mammalian enzyme which is
myristylated at the amino-terminal glycine. The lack of acylation did not
significantly alter the activity of the enzyme. The specific activity of 19
mumol/min/mg is comparable to the mammalian enzyme. The Km values for Kemptide
(Leu-Arg-Arg-Ala-Ser-Leu-Gly) (43 microM) and MgATP (18.5 microM also were
comparable. The absence of the acyl group also did not prevent holoenzyme
formation. Holoenzyme activation by cAMP was indistinguishable for holoenzyme
made with mammalian catalytic subunit and recombinant catalytic subunit. The
recombinant enzyme was more sensitive than the mammalian enzyme to heat
denaturation at 49 degrees C. The t1/2 for the recombinant catalytic subunit was
0.7 min in contrast to 3.9 min for the mammalian enzyme. This difference in
stability may be attributable to the lack of the acyl group. The recombinant
enzyme was particularly sensitive to heat denaturation in the presence of low
concentrations (0.01%) of Triton X-100.
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