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.
Hayden
M
M
Abbott Laboratories, Abbott Park, Illinois 60064-3500, USA.
Traphagen
L
L
Wilkins
J
J
Schmitz
E
E
Laird
D
D
Herrmann
R
R
Mandecki
W
W
D015415:Biomarkers; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D009206:Myocardium / Q000737:chemistry*; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014336:Troponin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D019210:Troponin I
D015415:Biomarkers; D011993:Recombinant Fusion Proteins; D014336:Troponin; D019210:Troponin I; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase