Characterization of the human calmodulin-like protein expressed in Escherichia coli

Biochemistry. 1992 Dec 29;31(51):12826-32. doi: 10.1021/bi00166a017.

Abstract

The protein-coding region of an intronless human calmodulin-like gene [Koller, M., & Strehler, E. E. (1988) FEBS Lett. 239, 121-128] has been inserted into a pKK233-2 expression vector, and the 148-residue, M(r) = 16,800 human protein was purified to apparent homogeneity by phenyl-Sepharose affinity chromatography from cultures of Escherichia coli JM105 transformed with the recombinant vector. Several milligrams of the purified protein were obtained from 1 L of bacterial culture. A number of properties of human CLP were compared to those of bacterially expressed human calmodulin (CaM) and of bovine brain CaM. CLP showed a characteristic Ca(2+)-dependent electrophoretic mobility shift on SDS-polyacrylamide gels, although the magnitude of this shift was smaller than that observed with CaM. CLP was able to activate the 3',5'-cyclic nucleotide phosphodiesterase to the same Vmax as normal CaM, albeit with a 7-fold higher Kact. In contrast, the erythrocyte plasma membrane Ca(2+)-ATPase could only be stimulated to 62% of its maximal CaM-dependent activity by CLP. CLP was found to contain four Ca(2+)-binding sites with a mean affinity constant of 10(5) M-1, a value about 10-fold lower than that for CaM under comparable conditions. The highly tissue-specifically-expressed CLP represents a novel human Ca(2+)-binding protein showing characteristics of a CaM isoform.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3',5'-Cyclic-AMP Phosphodiesterases / metabolism
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Calcium / metabolism
  • Calcium-Transporting ATPases / blood
  • Calmodulin / chemistry
  • Calmodulin / genetics*
  • Calmodulin / pharmacology
  • Cattle
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation / drug effects
  • Erythrocytes / enzymology
  • Escherichia coli / genetics*
  • Gene Expression*
  • Genetic Vectors
  • Humans
  • Molecular Sequence Data
  • Myocardium / enzymology
  • Osmolar Concentration
  • Plasmids
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / pharmacology
  • Transformation, Bacterial

Substances

  • Calmodulin
  • Recombinant Proteins
  • 3',5'-Cyclic-AMP Phosphodiesterases
  • Calcium-Transporting ATPases
  • Calcium