Overexpression of human cardiac troponin in Escherichia coli: its purification and characterization

Lohmann, K. and Westerdorf, Barbara and Maytum, Robin and Geeves, Michael A. and Jaquet, Kornelia (2001) Overexpression of human cardiac troponin in Escherichia coli: its purification and characterization. Protein Expression and Purification, 21 (1). pp. 49-59. ISSN 1046-5928 . (The full text of this publication is not available from this repository)

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Abstract

All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E. coli. The coexpressed subunits assembled within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC). The complex was isolated and purified by three chromatographic steps. Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3:cTnI:cTnC was obtained. Upon phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart. By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex.

Item Type: Article
Additional information: 1046-5928 (Print) Journal Article Research Support, Non-U.S. Gov't
Uncontrolled keywords: Amino Acid Sequence Animals Cattle Chromatography, Gel Cloning, Molecular/methods Cyclic AMP-Dependent Protein Kinases/metabolism Escherichia coli/genetics/metabolism Humans Mitochondria, Heart/chemistry Myocardium/*metabolism Nuclear Magnetic Resonance, Biomolecular Peptide Fragments/chemistry Phosphorylation Protein Binding Protein Subunits Recombinant Proteins/chemistry/isolation & purification/metabolism Serine Tropomyosin/chemistry/metabolism Troponin/*chemistry/isolation & purification/metabolism
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group
Depositing User: Michael Geeves
Date Deposited: 29 Aug 2008 11:10
Last Modified: 21 May 2014 07:36
Resource URI: http://kar.kent.ac.uk/id/eprint/3859 (The current URI for this page, for reference purposes)
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