TY - JOUR
T1 - Purification and functional analysis of protein kinase G-1α using a bacterial expression system
AU - Aggarwal, Saurabh
AU - Rafikov, Ruslan
AU - Gross, Christine M.
AU - Kumar, Sanjiv
AU - Pardo, Daniel
AU - Black, Stephen M.
N1 - Funding Information:
This research was supported in part by grants, HL60190 (to S.M.B.), HL67841 (to S.M.B.), HL084739 (to S.M.B.), R21HD057406 (to S.M.B.) all from the National Institutes of Health , by a grant from the Fondation Leducq (to S.M.B), a pre-doctoral fellowship from the Southeast Affiliates of the American Heart Association , 09PRE2400015 (to S.A.), and a Cardiovascular Discovery Institute Seed Award (to S.K.). Ruslan Rafikov was supported in part by NIH training Grant, 5T32HL06699 .
PY - 2011/10
Y1 - 2011/10
N2 - 3′,5′ Cyclic guanosine monophosphate (cGMP)-dependent protein kinase G-1α (PKG-1α) is an enzyme that is a target of several anti-hypertensive and erectile dysfunction drugs. Binding of cGMP to PKG-1α produces a conformational change that leads to enzyme activation. Activated PKG-1α performs important roles both in blood vessel vasodilation and in maintaining the smooth muscle cell in a differentiated contractile state. Recombinant PKG-1α has been expressed and purified using Sf9-insect cells. However, attempts at purifying full length protein in a soluble and active form in prokaryotes have thus far been unsuccessful. These attempts have been hampered by the lack of proper eukaryotic protein folding machinery in bacteria. In this study, we report the successful expression and purification of PKG-1α using a genetically engineered Escherichia coli strain, Rosetta-gami 2(DE3), transduced with full-length human PKG-1α cDNA containing a C-terminal histidine tag. PKG-1α was purified to homogeneity using sequential nickel affinity chromatography, gel filtration and ion exchange MonoQ columns. Protein identity was confirmed by immunoblot analysis. N-terminal sequencing using Edman degradation demonstrated that the purified protein was full length. Analysis of enzyme kinetics, using a nonlinear regression curve, identified that, at constant cGMP levels (10 μM) and varying ATP concentrations, PKG-1α had a maximal velocity (V max) of 5.02 ± 0.25 pmol/min/μg and a Michaelis-Menten constant (K m) of 11.78 ± 2.68 μM ATP. Recent studies have suggested that endothelial function can be attenuated by oxidative and/or nitrosative stress but the role of PKG-1α under these conditions is unclear. We found that PKG-1α enzyme activity was attenuated by exposure to the NO donor, spermine NONOate, hydrogen peroxide, and peroxynitrite but not by superoxide, suggesting that the attenuation of PKG-1α activity may be an under-appreciated mechanism underlying the development of endothelial dysfunction in a number of cardiovascular diseases.
AB - 3′,5′ Cyclic guanosine monophosphate (cGMP)-dependent protein kinase G-1α (PKG-1α) is an enzyme that is a target of several anti-hypertensive and erectile dysfunction drugs. Binding of cGMP to PKG-1α produces a conformational change that leads to enzyme activation. Activated PKG-1α performs important roles both in blood vessel vasodilation and in maintaining the smooth muscle cell in a differentiated contractile state. Recombinant PKG-1α has been expressed and purified using Sf9-insect cells. However, attempts at purifying full length protein in a soluble and active form in prokaryotes have thus far been unsuccessful. These attempts have been hampered by the lack of proper eukaryotic protein folding machinery in bacteria. In this study, we report the successful expression and purification of PKG-1α using a genetically engineered Escherichia coli strain, Rosetta-gami 2(DE3), transduced with full-length human PKG-1α cDNA containing a C-terminal histidine tag. PKG-1α was purified to homogeneity using sequential nickel affinity chromatography, gel filtration and ion exchange MonoQ columns. Protein identity was confirmed by immunoblot analysis. N-terminal sequencing using Edman degradation demonstrated that the purified protein was full length. Analysis of enzyme kinetics, using a nonlinear regression curve, identified that, at constant cGMP levels (10 μM) and varying ATP concentrations, PKG-1α had a maximal velocity (V max) of 5.02 ± 0.25 pmol/min/μg and a Michaelis-Menten constant (K m) of 11.78 ± 2.68 μM ATP. Recent studies have suggested that endothelial function can be attenuated by oxidative and/or nitrosative stress but the role of PKG-1α under these conditions is unclear. We found that PKG-1α enzyme activity was attenuated by exposure to the NO donor, spermine NONOate, hydrogen peroxide, and peroxynitrite but not by superoxide, suggesting that the attenuation of PKG-1α activity may be an under-appreciated mechanism underlying the development of endothelial dysfunction in a number of cardiovascular diseases.
KW - Cyclic GMP
KW - Protein kinase G
KW - Purification
KW - Rosetta-gami 2(DE3)
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UR - http://www.scopus.com/inward/citedby.url?scp=80051600602&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2011.05.001
DO - 10.1016/j.pep.2011.05.001
M3 - Article
C2 - 21600289
AN - SCOPUS:80051600602
SN - 1046-5928
VL - 79
SP - 271
EP - 276
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -
