EXPRESSION, PURIFICATION AND KINETIC-PROPERTIES OF HUMAN RECOMBINANT PHOSPHOLIPASE-C DELTA-3

To obtain sufficient quantities of pure phospholipase C delta 3 (PLC d elta 3) necessary for structural and kinetic studies, cDNA of human fi broblast PLC delta 3 was cloned in the pPROEX-1 vector, expressed in E . coli cells as a (6 x His) fusion protein and purified to homogeneity . From 1 L of E. coli culture 8 mg of pure PLC delta 3 was obtained by a two step purification procedure, which includes phosphocellulose an d Mono S cation exchange chromatography. The presence of His tag did n ot affect the catalytic and regulatory properties of PLC delta 3. The K-app for PIP2 was 142 +/- 11 and 156 +/- 12 mu M for His.PLC delta 3 and PLC delta 3, respectively. Recombinant PLC delta 3 showed an absol ute requirement for Ca2+. Increasing the free Ca2+ concentration from 0.2 to 0.5 mu M resulted in a sharp increase in enzyme activity. In co mparison with human recombinant PLC delta 1 the delta 3 isoenzyme was more sensitive to low Ca2+ concentration, The Ca2+ concentration yield ing maximal activation of PLC delta 1 and PLC delta 3 was 10 and 1 mu M, respectively. The activity of PLC delta 3 was stimulated by polyami nes and by basic proteins such as protamine, histone and mellitin. PLC delta 3 was activated most effectively by spermine and histone but th e extent of this activation was lower than for PLC delta 1.The data pr esented indicate that the expression of PLC delta 3 in E. coli cells p ermits to obtain active enzyme. The catalytic and regulatory propertie s of PLC delta 3 are similar to those of PLC delta 1.