Preparation And Functional Analysis Of Recombinant Human Soluble APRIL And Its Mutants

APRIL (a proliferation-inducing ligand) is a new member of the tumor necrosis factor (TNF) family, which is expressed in small amounts in normal tissues but produced abundantly in malignant tumors . It was recently shown that APRIL can not only promote malignant tumor growth in vivo and in vitro , but also prolong the life-span of the tumors. According to the close relationship between APRIL and malignant tumors , and the current status of the APRIL-inhibition molecules, we may develope a new strategy targeting against APRIL for tumor therapy based on APRIL mutants . So firstly in our experiment , the cDNA encoding the 105-250 amino acids of APRIL (soluble APRIL , sAPRIL) was cloned by RT-PCR and its two mutants were constructed by one-step opposite direction PCR. Secondly, the prokaryotic expression vectors containing the sAPRIL cDNA and its mutants respectively were transeformed into E.coli DH5αand induced by IPTG to express the corresponding proteins, which were purified by Ni2+-NTA chromatography. Thirdly, the functions of these purified proteins were identified. The results and conclusions are summarized as follows: 1. The cDNA of sAPRIL was cloned , which was consistent with the sequence encoding human APRIL105-250 amino acids (sAPRIL) reported in GenBank. 2. Two mutants of sAPRIL were successfully constructed by one-step opposite direction PCR . In one mutant (msAPRIL1) , the 187th Gln-encoding sequences was deleted, and in the other mutant (msAPRIL2) , it was taken placed by Gly-encoding sequences . 3. The recombinant vectors pQE-80L/sAPRIL , pQE-80L/msAPRIL1 and pQE-80L/ msAPRIL2 were successfully constructed , transformed into E.coli DH5αrespectively and expressed in the present of IPTG , and the three corresponding proteins were about 19 kDa identified by SDS-PAGE. 4. The expressed proteins were purified by Ni2+-NTA chromatography and refolded by dialysis against urea-buffer. SDS-PAGE analysis showed that the target proteins were highly purified. 5. The biological activities of the prepared proteins were analyzed by immunohisto-chemistry technique, FCM, MTT method and [3H]-Thymidine incorporation assay. The datas showed that the sAPRIL could strongly stimulate malignant tumor cell growth , while its mutans could bind to the receptors on these tumor cells and prevent their proliferation , even kill them and block the activity of sAPRIL in different extent . In summary , the functional recombinant human sAPRIL proteins and its mutans were successfully produced, which may pave a way for further study on the mechanism of APRIL and novel developing malignant tumor therapeutic agents based on APRIL mutants .