Expression in Escherichia coli and characterization of the heat-stable inhibitor of the cAMP-dependent protein kinase.
Pure heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) has been isolated in high yield by using a bacterial expression vector constructed to synthesize the complete sequence of the rabbit muscle protein kinase inhibitor, plus an amino-terminal initiator methionine and glycine. Bacterially expressed PKI has an inhibitory activity identical to that of the protein isolated from rabbit skeletal muscle and, by gel filtration and gel electrophoresis, has the same physicochemical characteristics as the native physiological form of PKI. Fourier transformed infrared spectroscopy and CD establish that PKI has unusually large amounts of random coil and turn structures, with significantly smaller amounts of alpha-helix and beta structures.
Thomas
J
J
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Van Patten
S M
SM
Howard
P
P
Day
K H
KH
Mitchell
R D
RD
Sosnick
T
T
Trewhella
J
J
Walsh
D A
DA
Maurer
R A
RA
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D047908:Intracellular Signaling Peptides and Proteins; D009132:Muscles / Q000201:enzymology; D010957:Plasmids; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D013053:Spectrophotometry
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C015482:protein kinase modulator