Expression in Escherichia coli and affinity purification of a CKS-troponin I fusion protein.

The human cardiac troponin I gene was subcloned and expressed at high levels in Escherichia coli as a fusion protein to CMP-KDO synthetase (CKS). Expression levels of the CKS-troponin I fusion were 8% of total cellular protein 4 h after induction with IPTG. The fusion was expressed primarily as an insoluble protein as shown by SDS-PAGE analysis. Expressed CKS-troponin I fusion from a crude lysate was antigenic against anti-CKS and anti-troponin I monoclonal antibodies in Western blots. The fusion was affinity-purified over a TnC affinity column using a urea-solubilized extract of a crude cell lysate. Serial dilutions of crude soluble extracts of the troponin I fusion were assayed in several microparticle enzyme immunoassays and found to exhibit similar immunogenic responses relative to cardiac troponin I isolated from human heart tissue.