Purification and characterization of phospholipase C-beta 1 mutants expressed in E. coli.
In the M1-muscarinic receptor-Gq-phospholipase C-beta 1 (PLC-beta 1) pathway, PLC-beta 1 is both the effector regulated by Gq and acts as GTPase activating protein (GAP) for Gq. To rapidly evaluate in vitro PLC-beta 1 mutants constructed by oligonucleotide-directed mutagenesis, we established a quick expression and purification procedure. A pQE60/His6PLC-beta 1 construct was expressed in E. coli SG13009[pREP4]. Purification (approximately 160-fold) was obtained after high-salt extraction and chromatography over Ni(+)-agarose, Mono Q and Mono S columns. Several His6PLC-beta 1 mutants were equally responsive to alpha q. GTP gamma S, although the mutant His6PLC-beta 1-P57 (G758D) had only 2.5% the intrinsic PLC activity of the wild type. Also, His6PLC-beta 1 wild type and mutants acted as GAPs for Gq in a reconstitution assay. Thus, the present procedure provides a method to quickly assess phospholipase activity, alpha q-responsiveness, and GAP activity of PLC-beta 1.
Meij
J T
JT
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
Ross
E M
EM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D054799:Phospholipase C beta; D011506:Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; D007527:Isoenzymes; D011506:Proteins; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins