Expression And Purification Of Recombinant Nematode Anticoagulant Peptide

Arterial and venous thrombosis are a major cause of morbidity and mortality.Currently available anticoagulants have undesirable properties that limit their usefulness in the treatment of thrombotic disorders and therefore novel anticoagulants are being sought. Nematode anticoagulant peptides (NAPs). a series of small peptides extracts from the hematophagous nematode Ancylostoma caninum, inhibit blood coagulation with picomolar inhibition constants, and have been identified as novel pharmaceutical agents.Object In the present work, we investigate the fusing expression in E.coli BL21(DE3), the purification and the activity detection of HIS-TRX- rNAP to lay the foundation for the development of a novel anticoagulant.Methods and Results1. Construction of pETlla/HIS-TRX-rNAP and expression of HIS-TRX-rNAPIn our previous study, we attempted to overexpress rNAP5 as a fusion protein linked to thioredoxin. To facilitate its purification, we expressed an N-terminal 8×His-tagged version of TRX-rNAP in E. coli BL21 (DE3) strain. The gene encoding HIS-TRX-rNAP was amplified by PCR and cloned into the prokaryotic expression vector pETlia between thesites of Ndel and BamHI, which was transformed into E.coli DH5a. The recombinant plasmid was confirmed by restriction enonulease analysis and DNA sequencing and was then transformed into E.coli BL21(DE3). The expression of the recombinant HIS-TRX-rNAP was induced by the addition of isopropylthio- ￡ -D-galactoside (IPTG) to the culture of transformed E.coli, and detected by SDS-PAGE and Western blot. The conditions for the fermentation process of the bacteria expressing HIS-TRX-rNAP including temperature, time, pH. IPTG and ampicillin concentration, etc. were optimized. Under these conditions, the yield in wet weight of cells was about 10 gram per liter of LB medium, and HIS-TRX-rNAP protein occupied approximately 30% of total bacterial protein.2. Purification and activity detection of fusion protein HIS-TRX-rNAPAfter fermentation, the cells were pelleted and resuspended. Following sonication and centrifugation, the supernatant containing the soluble proteins of the cell lysates was collected. After heating, anion exchange chromatography, Ni+-chelating chromatography and gel filtration chromatography, the recombiant HIS-TRX-rNAP was purified with a purity of over 98% and a recovery of over 50%. The purified protein was then respectively diluted to concentrations of 1 mg/ml, 0.5 mg/ml,0.1 mg/ml,0.05 mg/ml and 0.025 mg/ml in PBS and added to normal human plasma. The results showed that the recombiant fusion protein HIS-TRX-rNAP could prolong the PT/APTT of plasma compared with the PBS control and the effect was in a dose-dependent manner. These results demonstrate that HIS-TRX-rNAP has antithrombotic efficacy in Vitro.Conclusion The recombinant fusion protein HIS-TRX-rNAP was sucessfully over-expressed in E.coli BL21(DE3). The corresponding fermentation and purification...