Expression And Purification Of Netrophil Inhibitory Factor In E.coli

Neutrophil inhibitory factor(NIF)is a 41-kilodalton glycoprotein from the canine hookworm (Ancylostoma caninum) that binds CD11b/CD18 on neutrophil membrane. NIF comprises a mature polypeptide of 257 amino acids, preceded by a 17-amino acid leader. NIF is a novel anti-inflammatory agent, which blocks the adhesion of activated human neutrophils to vascular endothelial cells as well as the release of H2O2 from activated neutrophils . NIF can alleviate neutrophils-mediated tissue trauma.Objective: Pfizer Inc. in Amercia is conducting the studies on rNIF(recombinant neutrophil inhibitory factor) for treating reperfusion injury associated with ischemic stroke in a PhaseⅡclinical trial. Although Moyle et al. of Corvas International Inc in Amercia have reported that rNIF can be stably expressed in Pichia pastoris, it has not been reported that rNIF can be expressed in E.coli. The present study discussed expression in E.coli BL21(DE3)plysS and purification of rNIF. Methods: The cDNA encoding mature protein of NIF was amplified by PCR. After PCR product and expression vector pET-21a(+)were digested by NdeⅠand BamHⅠ, the cDNA was cloned into expression vector pET-21a(+) between the sites of NdeⅠand BamHⅠ, which was transformed into E.coli DH5α. The recombinant plasmid was determined, before it was transformed into E.coli BL21(DE3)plysS. After being induced with IPTG , E.coli BL21(DE3)plysS cell was collected and broken by ultrasonic processor. In the supernatant or precipitate of E.coli BL21(DE3)plysS lysates, a specific 28.9 kDa protein band appeared in 15％SDS-PAGE. Cell that expressed rNIF and medium were separated by centrifugation. The cell was in Tris-HCl buffer(pH8.0) and broken by ultrasonic processor. rNIF was purified by three step chromatography comprising of Q-Sepharose FF anion exchange , Hydroxyapatite ion exchange and Sephacryl S-100 gel filtration. HPLC analysis can determine purification of purified rNIF. The biological activity of rNIF was determined by neutrophil-plastic adhesion assay. Gel scanning of 15％SDS-PAGE can determine the rNIF percentage in the total cell protein. Results:The cDNA encoding mature protein of NIF was successfully amplified by PCR and cloned into expression vector pET-21a(+) between the sites of NdeⅠand BamHⅠ. Its cDNA sequence was determined. After three hour induction with IPTG, rNIF was over-expressed in E.coli BL21(DE3)plysS as inclusion bodies . After inclusion bodies of rNIF was renatured,renaturation solution was used to purify. It is indicated by Gelscanning of 15％SDS-PAGE that the expression level of rNIF was about 20％ of the total protein. It is indicated by HPLC analysis that rNIF was purified to approximately 98％. It is indicated by neutrophil-plastic adhesion assay that rNIF can efficiently inhibit the adhesion of neutrophils. Conclusion: rNIF can be over-expressed in E.coli BL21(DE3)plysS that has biological acivity in the present study.Moreover, a suit of corresponding purification methods for rNIF was obtained. These results show possibility that rNIF can be produced on a large scale using E.coli.