A new protocol for high-yield purification of recombinant human CXCL8((3-72))K11R/G31P expressed in Escherichia coli.
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.
Cheng
Hsi-Tsung
HT
Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan.
Huang
Kuo-Chun
KC
Yu
Hui-Yuan
HY
Gao
Kun-Jhih
KJ
Zhao
Xixing
X
Li
Fang
F
Town
Jennifer
J
Gordon
John R
JR
Cheng
Jya-Wei
JW
D002151:Calorimetry; D002634:Chemotaxis, Leukocyte; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000302:isolation & purification*; D009504:Neutrophils / Q000166:cytology; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D014461:Ultracentrifugation
C000604253:CXCL8(3-72)K11R,G31P, human; D016209:Interleukin-8; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins