Expression, Purification And Biological Activity Analyses Of Different Isoforms Of Recombinant Human Interluekin-8

Objective:As a multifunctional cytokine, interluekin-8(IL-8) belongs to the CXC subfamily of chemokines and is one of the major mediators in inflammatory responses. Instead of trandtional biochemical methods, there is no literature to report the utilization of protein fragment complementation assay (PCA) for biological activity measurement of IL-8.We try to achieve high level expression and efficient purification of recombinant human IL-8 (rhIL-8) and characterize its biological activity with the method of PCA.Methods:Three gene fragments encoding different isoforms of rhIL-8 were successfully expressed by cloned into the expression vectors pME-15-6His and pET-32a respectively and then transfected into mammalian cells HEK 293F or E. coli BL21(DE3). The IMAC affinity chromatography column was then used for the purification of all rhIL-8 isoforms. Biological activity analyses of the purified samples have also be performed in vivo using the Split-TEV GPCR activation assay, which is established based on the PCA method.Results:First, the eukaryotic expression vector pME-15-6His/IL-8 form I has been correctly constructed. The pre-IL-8 (residues 1-99) gene was successfully expressed in HEK 293F cells and the recombinant proteins were secreted by the cells into growth medium after removal their N-terminal signal peptides (residues 1-20). The resulting proteins have been purified and identified as the form I of rhIL-8 (residues 21-99). Second, the prokaryotic expression vectors pET-32a/IL-8 form II and pET-32a/IL-8 form III have been also constructed and the IL-8 genes were correctly expressed as N-terminal Trx-tagged proteins in E. coli. After purification and enterokinase cleavage, the resulting proteins have been demonstrated as the form II (residues 23-99) and form III (residues 28-99) of rhIL-8. Moreover, all three isoforms of purified rhIL-8 can bind and activate the receptor IL8RB with the EC50 values of 13.66,19.50 and 4.05 ng/ml respectively.Conclusion:Totally three isoforms of rhIL-8 were successfully cloned, expressed and purified in eukaryotic and prokaryotic systems with high biological activities. We believe this study will be valuable for facilitation of theoretical research and large-scale production of rhIL-8.