[High level expression of recombinant human sCD40L-IZ and identification of its biological activation].
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.
Zhou
Xuan
X
Stem Cell Research Laboratory of Jiangsu Province, Medical Biotechnology Institute, Soochow University, Suzhou 215007, China. smbxuegz@public1.sz.js.cn
Yu
Jian-Feng
JF
Xu
Ying
Y
Qi
Chun-Jian
CJ
Huang
Yong
Y
Wang
Yong
Y
Zhang
Xue-Guang
XG
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*
D019013:CD40 Antigens; D011993:Recombinant Fusion Proteins; D023201:CD40 Ligand