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

Abstract Phospholipase C isoforms δ 1 and δ 3 (PLCδ 1 and δ 3 ) were expressed in Escherichia coli using the cDNA sequences from human fibroblasts. The enzymes were also expressed with the sequence Met-Gly-His 6 -Ser-Gly-Leu-Phe-Lys-Arg, a hexahistidine sequence followed by a Kex2 protease cleavage site, denoted as “-H6K2,” attached to their amino termini. PLCδ 1 , PLCδ 1 -H6K2, PLCδ 3 , and PLCδ 3 -H6K2 all expressed in highly active form. The H6K2-bearing isoforms were each purified to homogeneity in a single step, with yields of 90–100%, using agarose–iminodiacetic acid–Ni columns and imidazole buffer as eluting agent. Yields in terms of activity increased as the temperature of expression was decreased. Expression at 16°C for 72 h yielded 33 mg of pure PLCδ 1 -H6K2 and 13 mg of pure PLCδ 3 -H6K2 per liter of culture. Removal of H6K2 from both isoforms with Kex2 protease resulted in little or no loss of activity. Expression of PLC isoforms bearing -H6K2 at the amino terminus resulted in about 12 times more activity than expression of the isoforms lacking -H6K2. PLCδ 3 is much less stable than PLCδ 1 . Successful purification and storage of PLCδ 3 depends on a suitable stabilizing medium. Both isoforms require 0.3 μ m calcium ion for half-maximum activity. The specific activities of the isoforms expressed with and without -H6K2 are the same, as are their calcium saturation curves.