Meij J T, Ross E M
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
Biochem Biophys Res Commun. 1996 Aug 23;225(3):705-11. doi: 10.1006/bbrc.1996.1239.
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.
