Expression of cGMP-dependent protein kinase in Escherichia coli.
Cyclic GMP-dependent protein kinase (cGMP kinase) is involved in the relaxation of smooth muscle. The enzyme has been cloned and expressed in eukaryotic cell lines but so far not in prokaryotic cells. Three vectors were constructed for the expression of I alpha cGMP kinase in Escherichia coli. Transformation with the pET3a/cgk vector which uses the T7 RNA polymerase/promotor system resulted in efficient accumulation of cGMP kinase. Most of the protein was in an insoluble and catalytic inactive form. Various solubilization and refolding conditions did not yield an active enzyme. A small fraction of the cGMP kinase was present in the soluble cell extract. This fraction bound cGMP with high affinity but had no cGMP stimulated kinase activity. To prevent aggregation two additional vectors were constructed. (I) A bacterial leader sequence, which directs the export of proteins into the periplasmic space, was fused to the amino-terminus of the cGMP kinase. (II) A gram/gram+ shuttle vector for expression under the control of the tac promotor was used. Both constructs directed the synthesis of an insoluble and inactive cGMP kinase. These results suggest that large amounts of cGMP kinase can be expressed in E. coli, but mainly in an insoluble and inactive form. In contrast to eukaryotic cells, bacteria may lack systems for correct protein folding and/or posttranslational modification that are crucial for the productive folding and/or activation of cGMP kinase.
Feil
R
R
Institut für Pharmakologie und Toxikologie, Technische Universität München, Germany.
Bigl
M
M
Ruth
P
P
Hofmann
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D017869:Cyclic GMP-Dependent Protein Kinases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000254:growth & development; D005822:Genetic Vectors; D015151:Immunoblotting; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology; D009842:Oligopeptides / Q000138:chemical synthesis; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D009842:Oligopeptides; D011994:Recombinant Proteins; D017869:Cyclic GMP-Dependent Protein Kinases