In the M1-muscarinic receptor-G(q)-phospholipase C-β1 (PLC-β1) pathway, PLC-β1 is both the effector regulated by G(q) and acts as GTPase activating protein (GAP) for G(q). To rapidly evaluate in vitro PLC-β1 mutants constructed by oligonucleotide-directed mutagenesis, we established a quick expression and purification procedure. A pQE60/His6PLC-β1 construct was expressed in E. coli SG13009[pREP4]. Purification (~ 160-fold) was obtained after high-salt extraction and chromatography over Ni+-agarose, Mono Q and Mono S columns. Several His6PLC-β1 mutants were equally responsive to α(q)·GTPγS, although the mutant His6PLC-β1-P57 (G758D) had only 2.5% the intrinsic PLC activity of the wild type. Also, His6PLC-β1 wild type and mutants acted as GAPs for G(q) in a reconstitution assay. Thus, the present procedure provides a method to quickly assess phospholipase activity, αq-responsiveness, and GAP activity of PLC-β1.
|Original language||English (US)|
|Number of pages||7|
|Journal||Biochemical and Biophysical Research Communications|
|State||Published - Aug 23 1996|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology