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

• Published in: Biochemistry (Easton) Vol. 31; no. 51; pp. 12826 - 12832
• Main Authors: Rhyner, Johannes A, Koller, Markus, Durussel-Gerber, Isabelle, Cox, Jos A, Strehler, Emanuel E
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 29.12.1992
• Subjects: 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Calcium - metabolism; calcium-binding protein; Calcium-Transporting ATPases - blood; calmodulin; Calmodulin - chemistry; Calmodulin - genetics; Calmodulin - pharmacology; Cattle; characterization; Electrophoresis, Polyacrylamide Gel; Enzyme Activation - drug effects; Erythrocytes - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Vectors; Humans; like; man; Molecular Sequence Data; Myocardium - enzymology; Osmolar Concentration; Plasmids; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - pharmacology; Transformation, Bacterial; Human; Heterologous system; Calcium; Escherichia coli; Molecular interaction; Gene expression; In vitro; Biological activity; Binding protein; Gene; Analog; Bacteria; Recombinant protein; Calmodulin; Enterobacteriaceae
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00166a017

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.