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[High level expression of recombinant human sCD40L-IZ and identification of its biological activation]
Xuan Zhou1, Jian-Feng Yu, Ying Xu
1Stem Cell Research Laboratory of Jiangsu Province, Medical Biotechnology Institute, Soochow University, Suzhou 215007, China. smbxuegz@public1.sz.js.cn
Aim:
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.
Conclusion:
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.
