Baculovirus expression and purification of the second messenger enzyme phospholipase C-gamma 1, a tyrosine kinase substrate.
A rat cDNA encoding phospholipase C-gamma 1 (PLC-gamma 1) was expressed as a histidine-tagged fusion protein in insect cells utilizing the expression vector pBlueBacHis. The fusion protein was purified by Ni(2+)-agarose affinity chromatography to apparent homogeneity as defined by SDS-PAGE and Coomassie staining. Using a Triton X-100/PIP2 mixed micelle assay, (His6)-PLC-gamma 1 exhibited a calcium-dependent specific enzyme activity of 3 mumol/min/mg, a value similar to that reported for purified bovine brain PLC-gamma 1. Also similar to bovine brain PLC-gamma 1, (His6)-PLC-gamma 1 activity was stimulated by phosphatidic acid and inhibited by adenosine 5'monophosphate. (His6)-PLC-gamma 1 interacted directly with two known PLC-gamma 1 binding proteins, dynamin and the activated EGF receptor. Also, purified (His6)-PLC-gamma 1 was a tyrosine phosphorylation substrate for purified EGF receptor. These results suggest that (His6)-PLC-gamma 1 can be overexpressed as a functional enzyme in baculovirus-infected insect cells and purified by a one-step metal affinity chromatography procedure.
Horstman
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DA
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Ball
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R
Carpenter
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D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D006639:Histidine; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015290:Second Messenger Systems; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma