Cloning, expression, and purification of soluble human interleukin-4 receptor in Streptomyces.
Interleukin-4 (IL-4) is a pleiotropic cytokine which plays a pivotal role in shaping immune responses and mediating important proinflammatory functions in asthma. This cytokine exerts its biological effects through binding to its receptor (IL-4R) complex, with the alpha chain as the high-affinity binding subunit. Soluble IL-4R (sIL-4R) lacks the transmembrane and cytoplasmic domains, so it cannot induce cellular activation. By acting as a decoy to circulating IL-4 and neutralizing its activity, the high specificity and affinity of sIL-4R make it ideal as an IL-4 antagonist. In this study, a sIL-4R cDNA encoding the extracellular domain of IL-4R alpha chain was cloned into a Streptomyces-Escherichia coli shuttle plasmid pSGLgpp and expressed secretly in Streptomyces lividans TK 24. On SDS-PAGE gel, the expressed sIL-4R protein showed a Mw of 24 kDa, agreeable with the predicted size. The N-terminal sequence of the protein was also determined, confirming its identity and indicating that no degradation occurred at the N-terminus. With DEAE-Sepharose Fast Flow and Superdex HR 75 columns, the protein was purified and used on HPLC analysis, the purity reaching about 90%. The results of the ligand-binding blot and ELISA showed that such protein has biological activity of binding with ligand IL-4.
Zhang
Yong
Y
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical College, Tiantan Xili 1, Beijing 100050, PR China. zhangyong7599@yahoo.com.cn
Wang
Wei Chi
WC
Li
Yuan
Y
D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D015847:Interleukin-4 / Q000378:metabolism; D008024:Ligands; D019948:Receptors, Interleukin-4 / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D048170:Streptomyces lividans / Q000378:metabolism*
D008024:Ligands; D019948:Receptors, Interleukin-4; D011994:Recombinant Proteins; D015847:Interleukin-4