A new protocol for high-yield purification of recombinant human CXCL8((3-72))K11R/G31P expressed in Escherichia coli.

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, we have successfully constructed and expressed hG31P in Escherichia coli. Moreover, we have developed 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. The purity of hG31P is more than 95% and the final yield is 9.7mg hG31P per gram of cell paste. The purified hG31P was tested by various biological assays. In addition, the structural properties of hG31P were studied by circular dichroism (CD), ultracentrifuge, isothermal titration calorimetry (ITC), and nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that this purification protocol is very simple and easy to amplify at a large scale. The results of this study will provide an effective route to produce enough hG31P for future clinical studies.

journal_name

Protein Expr Purif

authors

Cheng HT,Huang KC,Yu HY,Gao KJ,Zhao X,Li F,Town J,Gordon JR,Cheng JW

doi

10.1016/j.pep.2008.04.007

subject

Has Abstract

pub_date

2008-09-01 00:00:00

pages

65-72

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(08)00117-4

journal_volume

61

pub_type

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