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 express ed primarily as an insoluble protein as shown by SDS-PAGE analysis. Ex pressed CKS-troponin I fusion from a crude lysate was antigenic agains t anti-CKS and anti-troponin I monoclonal antibodies in Western blots. The fusion was affinity-purified over a TnC affinity column using a u rea-solubilized extract of a crude cell lysate. Serial dilutions of cr ude soluble extracts of the troponin I fusion were assayed in several microparticle enzyme immunoassays and found to exhibit similar immunog enic responses relative to cardiac troponin I isolated from human hear t tissue. (C) 1995 Academic Press, Inc.