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Expression, purification, and characterization of the cGMP-dependent protein kinases I beta and II using the baculovirus system.

FEBS letters (1995-11-06)
D Pöhler, E Butt, J Meissner, S Müller, M Lohse, U Walter, S M Lohmann, T Jarchau
ABSTRACT

Detailed studies of differences in distinct cGMP kinase isoforms are highly dependent on expression of large amounts of these enzyme isoforms that are not easily purified by conventional methods. Here cGMP-dependent protein kinases, the type I beta soluble form from human placenta, and the type II membrane-associated form from rat intestine, were each expressed in a baculovirus/Sf9 cell system and purified in milligram amounts by affinity chromatography. The expressed recombinant proteins displayed characteristics like those of their native counterparts. cGK I beta was expressed as a 76 kDa protein predominantly found in the cytosol fraction, whereas cGK II was expressed as an 86 kDa protein predominantly associated with the membrane fraction. The apparent Ka and Vmax of cGMP for activation of cGK I beta were 0.5 microM and 3.4 mumol/min/mg, and for cGK II were 0.04 microM and 1.8 mumol/min/mg.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
PKG1alpha active human, recombinant, expressed in baculovirus infected Sf9 cells, ≥70% (SDS-PAGE)
Sigma-Aldrich
Protein Kinase G Iβ human, ≥95% (SDS-PAGE), recombinant, expressed in baculovirus infected Sf9 cells, buffered aqueous glycerol solution
Sigma-Aldrich
Protein Kinase G II from rat, >90% (SDS-PAGE), recombinant, expressed in baculovirus infected Sf9 cells, solution

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