Clonación, expresión y purificación de la troponina I cardiaca humana y su fragmento aminoterminal en Escherichia coli

Cloning, Expression and Purification of Human Cardiac Troponin I and Its Specific Amino-terminal Fragment in Escherichia coli. The human cardiac troponin I (TnIc) is nowadays the most cardiospecific diagnostic marker for acute myocardial infarction. The cardiac isoform of troponin I contains 32 additional amino acids on the Nterminus, which confer cardiac specificity. This paper describes the isolation of TnIc gen through reverse transcriptionpolymerase chain reaction using total RNA from cardiac tissue, and its cloning into an expression vector. The cardiospecific aminoterminal fragment of TnIc was cloned into the same expression vector. These molecules were obtained to high levels in Escherichia coli, representing 43% and 30% of total cellular protein, respectively. Both molecules were highly expressed as insoluble proteins. The entire protein and its fragment were purified by electroelution with a 94% and 92% purity, respectively. The yields of recombinant cardiac troponin I and recombinant aminoterminal fragment of TnIc were 41 mg and 23 mg per liter of E. coli culture, respectively. The recombinant TnIc and the fragment showed antigenic properties similar to those of the natural TnIc in ultramicroELISA. These molecules are thus good immunogens for obtaining anti-TnIc cardiospecific monoclonal antibodies, which are very important for the development of diagnostic kits for detecting acute myocardial infarction.