Phospholipase C isoforms delta 1 and delta 3 from human fibroblasts. High-yield expression in Escherichia coli, simple purification, and properties.
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 Met-Gly-His6-Ser-Gly-Leu-Phe-Lys-Arg, a hexahistidine sequence followed by a Kex2 protease cleavage site, denoted as "-H6K2," attached to their amino termini. PLC delta 1, PLC delta 1-H6K2, PLC delta 3, PLC delta 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 degrees C for 72 h yielded 33 mg of pure PLC delta 1-H6K2 and 13 mg of pure PLC delta 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. PLC delta 3 is much less stable than PLC delta1. Successful purification and storage of PLC delta 3 depends on a suitable stabilizing medium. Both isoforms require 0.3 microM 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.
Ghosh
S
S
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254, USA.
Pawelczyk
T
T
Lowenstein
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D054803:Phospholipase C delta; D043484:Proprotein Convertases; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins; D013696:Temperature; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D007527:Isoenzymes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D010738:Type C Phospholipases; C518024:PLCD1 protein, human; C518028:PLCD3 protein, human; D054803:Phospholipase C delta; C518026:Plcd1 protein, rat; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae