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Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:7108505
;
The cDNA for the bovine type I regulatory subunit of cAMP-dependent protein kinase has been inserted into the expression vector pUC7. When E. coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 2-4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-N/sub 3/-(/sup 32/P)-cAMP following transfer from SDS polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of IPTG. R-subunit accumulated in large amounts only in the stationary phase of growth. The addition of IPTG during the log phase of growth actually blocked the accumulation of R-subunit. The soluble, dimeric R-subunit was purifided to homogeneity by affinity chromatography. This R-subunit bound 2 mol cAMP/mol R monomer, reassociated with C-subunit to form cAMP-dependent holoenzyme, and migrated as a dimer on SDS polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties the expressed protein was indistinguishable from R/sup I/ purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z gene of the vector. The NH/sub 2/-terminal sequence was confirmed by amino acid sequencing.
Research Organization:
Univ. of California, San Diego, La Jolla
OSTI ID:
7108505
Report Number(s):
CONF-8606151-
Conference Information:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
550701 -- Microbiology-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
AMP
ANIMALS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CATTLE
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DNA
DOMESTIC ANIMALS
ELECTROPHORESIS
ENZYMES
ESCHERICHIA COLI
GROWTH
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MICROORGANISMS
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PROTEIN STRUCTURE
PROTEINS
PROTEOLYSIS
RADIOISOTOPES
RUMINANTS
SEPARATION PROCESSES
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES