PHOSPHOLIPASE-C ISOFORMS DELTA(1) AND DELTA(3) FROM HUMAN FIBROBLASTS- HIGH-YIELD EXPRESSION IN ESCHERICHIA-COLI, SIMPLE PURIFICATION, ANDPROPERTIES

Phospholipase C isoforms delta(1) and delta(3) (PLC delta(1) and delta (3)) were expressed in Escherichia coli using the cDNA sequences from human fibroblasts. The enzymes were also expressed with the sequence M et-Gly-His(6)-Ser-Gly-Leu-Phe-Lys-Arg, a hexahistidine sequence follow ed by a Kex2 protease cleavage site, denoted as ''-H6K2,'' attached to their amino termini. PLC delta(1), PLC delta(1)-H6K2, PLC delta(3), a nd PLC delta(3)-H6K2 all expressed in highly active form. The H6K2-bea ring isoforms were each purified to homogeneity in a single step, with yields of 90-100%, using agarose-iminodiacetic acid-Ni columns and im idazole buffer as eluting agent, Yields in terms of activity increased as the temperature of expression was decreased, Expression at 16 degr ees C for 72 h yielded 33 mg of pure PLC delta(1)-H6K2 and 13 mg of pu re PLC delta(3)-H6K2 per liter of culture, Removal of H6K2 from both i soforms with Kex2 protease resulted in little or no loss of activity, Expression of PLC isoforms-bearing -H6K2 at the amino terminus resulte d in about 12 times more activity than expression of the isoforms lack ing -H6K2. PLC delta(3) is much less stable than PLC delta(1). Success ful purification and storage of PLC delta(3) depends on a suitable sta bilizing medium. Both isoforms require 0.3 mu M calcium ion for half-m aximum activity, The specific activities of the isoforms expressed wit h and without -H6K2 are the same, as are their calcium saturation curv es. (C) 1997 Academic Press.