title: [High level expression of recombinant human sCD40L-IZ and identification of its biological activation].
abstract: OBJECTIVE
  To clone and express the recombinant human soluble CD40 ligand(CD40L) with biological activation.
  
  
  METHODS
  The isoleucine zipper (IZ) gene was fused at N-terminal of CD40L gene coding E107-L261 contributing to form trimer. The gene coding hexahistidine was fused at the N-terminal of IZ because the sCD40L-IZ fusion protein could be purified by affinity chromatography. Then the fusion gene was amplified and cloned into expression plasmid pET30a.
  
  
  RESULTS
  The protein sCD40L-IZ with His-tag at the N-terminal was effectively expressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to acquire soluble sCD40L-IZ. The fusion protein sCD40L-IZ was conveniently purified using HiTrap affinity column with above 95% purity. The gel filtration chromatography and non-reduced SDS-PAGE identified the trimeric structure of the recombinant protein. The microscope analysis showed the sCD40L-IZ interacted with the membrane CD40 on XG2, a multiple myeloma cell line.
  
  
  CONCLUSIONS
  The recombinant human trimeric CD40L-IZ was expressed in prokaryotic cells successfully, which has provided a basis for further study of the relationship between CD40L and apoptosis, CD40L and pathogenesis of illness. It is also useful to clinical treatment.
authors.affiliation: Stem Cell Research Laboratory of Jiangsu Province, Medical Biotechnology Institute, Soochow University, Suzhou 215007, China. smbxuegz@public1.sz.js.cn
authors.forename: Xuan
authors.initials: X
authors.lastname: Zhou
authors.forename: Jian-Feng
authors.initials: JF
authors.lastname: Yu
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.forename: Chun-Jian
authors.initials: CJ
authors.lastname: Qi
authors.forename: Yong
authors.initials: Y
authors.lastname: Huang
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.forename: Xue-Guang
authors.initials: XG
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D019013:CD40 Antigens / Q000378:metabolism; D023201:CD40 Ligand / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
chemical_list: D019013:CD40 Antigens; D011993:Recombinant Fusion Proteins; D023201:CD40 Ligand