Abstract
The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8) binds to both the CXCR1 and CXCR2 receptors with high affinity and the expression levels of CXCL8 are elevated in many inflammatory diseases. Recently, an analogue of human CXCL8, CXCL8(3–72)K11R/G31P (hG31P) has, been developed. It has been demonstrated that hG31P is a high affinity antagonist for both CXCR1 and CXCR2. To obtain large quantities of hG31P, this study has successfully constructed and expressed hG31P in Escherichia coli. Moreover, a new protocol for high-yield purification of hG31P and for the removal of lipopolysaccharide (LPS, endotoxin) associated with hG31P due to the expression in E. coli has been developed. The solution structure of hG31P is also demonstrated in this study. In addition, the structural properties of hG31P were studied by circular dichroism (CD), ultracentrifuge, isothermal titration calorimetry (ITC), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy. The results demonstrated that the possible mechanism on hG31P to inhibit signal transduction is contributed from the perturbation of the G31P loop. Furthermore, this study also indicated that this purification protocol is very simple and easy to amplify at a large scale. The results of this study will provide good explanation for the antagonist according to the structure basis, and it also provides effective route to produce large quantities of hG31P for future clinical studies.