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Expression of cGMP-dependent protein kinase inEscherichia coli (1993)

Feil, R. ; Bigl, M. ; Ruth, P. ; [et al.]

Springer

Molecular and cellular biochemistry 127-128 (1993), S. 71-80

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ISSN: 1573-4919

Keywords: Cyclic GMP-dependent protein kinase ; Escherichia coli ; post-translational modification ; protein folding

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: Abstract 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 Iα cGMP kinase inEscherichia 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 aminoterminus 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 isoluble and inactive cGMP kinase. These results suggest that large amounts of cGMP kinase can be expressed inE. coli, but mainly in an isoluble 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.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01076758

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Enhanced chemiluminescent immunoassay for aldosterone (1990)

Hubl, W. ; Thorpe, G. H. G. ; Hofmann, F. ; [et al.]

New York : Wiley-Blackwell

Journal of Bioluminescence and Chemiluminescence 5 (1990), S. 49-52

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ISSN: 0884-3996

Keywords: Aldosterone ; enhanced chemiluminescence ; immunoassay ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology

Notes: A solid phase immunoassay for aldosterone using enhanced chemiluminescent detection has been developed. Monoclonal antibodies against aldosterone were used for the immune reaction and compared with polyclonal antibodies. Uniform Protein A coated polystyrene tubes were used as solid phase for the monoclonal antibody and second (anti-rabbit) antibody coated tubes for the polyclonal antibody. Horseradish peroxidase was covalently linked to aldosterone as enzyme label. Optimum conditions were established for the generation and measurement of the luminescent reactions using luminol, p-iodophenol as enhancer and hydrogen peroxide.The advantages of this assay are the high sensitivity with a detection limit of 100fg/tube, the prolonged luminescence signal with a simplification of the measurement (simpler detectors, external start pipetting) and the short measure time with the possibility of repeated measurement. The coefficients of variation were 4.2%-7.3% in the concentration range 140-1180 pmol/l. The assay showed a significant correlation (r = 0.91) with the ELISA.The aldosterone concentrations in plasma and saliva of patients with Conn's syndrome were significantly increased, and in patients with Addison's disease were found near the detection limit.

Additional Material: 1 Ill.