TY - JOUR
T1 - Purification and characterization of phospholipase C-β1 mutants expressed in E. coli
AU - Meij, Johanna T A
AU - Ross, Elliott M.
N1 - Funding Information:
The authors wish to thank Gloria Biddlecome and Dr. Ruth H. Paulssen for providing purified αq and for conducting the GTPase assays. J.T.A. Meij was an International Research Fellow of the American Heart Association. This work was supported by NIH grant GM30355 and R.A. Welch Foundation grant I-0982.
PY - 1996/8/23
Y1 - 1996/8/23
N2 - 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.
AB - 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.
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U2 - 10.1006/bbrc.1996.1239
DO - 10.1006/bbrc.1996.1239
M3 - Article
C2 - 8780678
AN - SCOPUS:0030598832
SN - 0006-291X
VL - 225
SP - 705
EP - 711
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -