ABSTRACT
The novel calcineurin inhibitor, dibefurin, has been isolated from the fungal culture AB 1650I-759. The isolation was bioactivity-directed fractionation using an assay which measures the phosphatase activity of calcineurin. The compound was purified by countercurrent, reverse phase and gel filtration chromatographies. Several studies, including crystallographic, NMR and MS, revealed that dibefurin is a novel dimeric compound of a unique structural type.
Subject(s)
Benzofurans/pharmacology , Calmodulin-Binding Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Fungi/metabolism , Phosphoprotein Phosphatases/antagonists & inhibitors , Benzofurans/chemistry , Calcineurin , Chromatography, Gel , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Fermentation , Magnetic Resonance Spectroscopy , Molecular Structure , Spectroscopy, Fourier Transform InfraredABSTRACT
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.
