Purification and functional analysis of protein kinase G-1α using a bacterial expression system

Saurabh Aggarwal, Ruslan Rafikov, Christine M. Gross, Sanjiv Kumar, Daniel Pardo, Stephen Matthew Black

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)271-276
Number of pages6
JournalProtein Expression and Purification
Volume79
Issue number2
DOIs
StatePublished - Oct 1 2011

Fingerprint

Cyclic GMP-Dependent Protein Kinases
Cyclic GMP
Enzymes
Adenosine Triphosphate
Guanosine Monophosphate
Sf9 Cells
Proteins
Peroxynitrous Acid
Enzyme Activation
Ion Exchange
Protein Folding
Erectile Dysfunction
Heat-Shock Proteins
Nickel
Affinity Chromatography
Recombinant Proteins
Histidine
Vasodilation
Superoxides
Hydrogen Peroxide

Keywords

  • Cyclic GMP
  • Protein kinase G
  • Purification
  • Rosetta-gami 2(DE3)

ASJC Scopus subject areas

  • Biotechnology

Cite this

Purification and functional analysis of protein kinase G-1α using a bacterial expression system. / Aggarwal, Saurabh; Rafikov, Ruslan; Gross, Christine M.; Kumar, Sanjiv; Pardo, Daniel; Black, Stephen Matthew.

In: Protein Expression and Purification, Vol. 79, No. 2, 01.10.2011, p. 271-276.

Research output: Contribution to journalArticle

Aggarwal, Saurabh ; Rafikov, Ruslan ; Gross, Christine M. ; Kumar, Sanjiv ; Pardo, Daniel ; Black, Stephen Matthew. / Purification and functional analysis of protein kinase G-1α using a bacterial expression system. In: Protein Expression and Purification. 2011 ; Vol. 79, No. 2. pp. 271-276.
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