CALCIUM-BINDING BY CHICK CALRETININ AND RAT CALBINDIN-D28K SYNTHESIZED IN BACTERIA

Calretinin is a member of the EF-hand calcium-binding protein family, with a high similarity with calbindin D28k. The chick calretinin cDNA sequence was reconstructed in a M13 vector and transferred into an exp ression plasmid derived from the pET series. The calretinin gene was e xpressed in Escherichia coli and produced immunoreactive calretinin of the expected size. Bacterially expressed calretinin was purified with successive ammonium-sulfate precipitation, DEAE chromatography, hydro xyapatite chromatography, Sephadex G-75 chromatography and Mono-Q chro matography. Normally, 1.0-1.5 mg calretinin was obtained from 1 l bact erial culture with a protein recovery of 0.5-1.5 %. Calbindin D28k was purified similarly from bacteria using an expression plasmid provided by W. Hunziker. Calcium-binding activity of purified proteins was mea sured by equilibrium dialysis in calcium/EGTA mixtures with Ca-45 as t racer. Both calretinin and calbindin D28k bound 3-4 Ca2+/molecule (cal retinin, 4.0 +/- 0.5; calbindin D28k, 3.5 +/- 0.4), implying that at l east one of the canonical EF-hand domains does not bind calcium. The K (d) was 0.3-0.5 muM with little difference between the values for the two proteins.