High-level expression of complementary DNA encoding rat calmodulin in Escherichia coli.
We report the production and characterization of a rat calmodulin made in Escherichia coli. To express the rat calmodulin cDNA in E. coli, we have employed an expression vector containing the E. coli trp promoter and trpA terminator. The cDNA was modified so as to delete the 5' nontranslated sequence and to incorporate a consensus sequence for the E. coli ribosome-binding site. Several codons for the N-terminal amino acids were selected to fit the E. coli consensus nucleotide sequence around the translational initiation codon. After induction of expression in E. coli, rat calmodulin accounted for over 30% of total cellular proteins. About 100 mg of recombinant rat calmodulin, purified to over 90% homogeneity by extraction from bacterial lysate followed by phenyl-Sepharose column chromatography, was obtained from 1 liter of E. coli culture. This recombinant calmodulin activated rat brain cyclic AMP phosphodiesterase to the same extent as the native calmodulin purified from rat brain. These results indicate that the overproduction system of the recombinant calmodulin in E. coli facilitates the study of the structure-function relationship by site-specific mutagenesis.
Kirin Brewery Company, Ltd., Pharmaceutical Laboratory, Gunma, Japan.
S
S
Matsuki
T
T
Ozawa
S
S
Nagao
H
H
Hirata
H
H
Kanoh
Y
Y
Nozawa
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002147:Calmodulin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D002147:Calmodulin; D011994:Recombinant Proteins; D004247:DNA