The Cloning, Expression And Biological Function Of Recombinant Human TALL-1

The Human Genome Project is forcing genomics to become the leader of medical science. As new discoveries in this arena are applied, companies and industries are being restructured in a way that will change the world's economy. In this era the research about genome based drug have great potential. At least 19 TNF ligands and 29 receptors have been identified so far. As a group of important cytokine in mammalian cells they play pivotal roles in many biological processes, such as in host defense, inflammation, apoptosis, autoimmunity and organogenesis.The novel TNF family member TALL-1 (TNF- and ApoL-related leukocyte expressed ligand 1), also known as BlyS, THANK, BAFF, zTNF4 and TNFSF20, has been identified by various groups in 1999. Studied of TALL-1 have revealed high levels of expression in peripheral blood mononuclear cells (PBMCs), lymph node, spleen, bone marrow and so on. TALL-1 exists as a full-length 285-amino acid, type II transmembrane protein as well as a soluble protein (amino acids 134-285) derived from the membrane-bound form by cleavage with a putative furin family protease. The extracellular domain of TALL-1 shows highest sequence homology with APRIL, and lesser homology with TNF, FasL, LT, TRAIL, LT a LIGHT and RANKL. TALL-1 binds to three receptors: B-cell maturation protein (BCMA), transmembrane activator and CAML interactor (TACI) and BAFF receptor (BAFF-R). TALL-1 can initiate diverse biological functions depending on the receptor expression profile of target cells. Firstly, as an important stimulatory and survival factor for B cells, TALL-1 is essential for B cell survival and maturation. Secondly, studies of TALL-1 have revealed it can regulate T cell activation, with an overall enhancement of proliferation and effector's responses. Thirdly, TALL-1 can strongly suppressed the growth of tumor cell lines, which is similar to other TNF ligands. Fourthly, transgenic mice overexpressing TALL-1 have a vast increase in mature B cells, enlarged spleens, high plasma cell numbers, and high levels of autoantibodies. They thus display a syndrome resembling a human autoimmune disease.Whereas TALL-1 has showed pleiotropic biological functions, it soon becomes a promising new star in the field of life science. To explore its bioactivity and lay foundations for further research in the possibility of clinical trial, we plan to clone, express TALL-1 protein and construct its mutant. The experiments were performed as below:1. Human TALL-1 cDNA and the TALL-1 extracellular domain cDNA was amplified with total RNA from human lymph node tissue by RT-PCR, then cloned into Pichia pastoris expression vector pPIC-9K. The cloned human TALL-1 cDNA and the TALL-1 extracellular domain cDNA sequence are identical to the published sequence and the gene's expression was under the alcohol oxidase (A0X1) promoter control and the coding sequence was fused to the alpha-mating factor signal by sequence.2. The plasmid pPIC-9K-hTALL-l/sTALL-l was electrotransformed into the yeast strain GS115 after being linearized by Sad. The high copy member of heterogenes was screened from the positive engineering yeast transformants by PCR and G418 and expressed in Pichia pastoris under the induction of methanol. We analyzed the expressed products by RT-PCR, 3'RACE, SDS-PAGE, Western blot, ELISA and MTT cell proliferation assay after the expression condition was optimized. Our results demonstrated that the expressed protein with good antigen specificity exist in the supernatant of the culture and can inhibit the growth of Hela.3. The human TALL-1 cDNA and the TALL-1 extracellular domain cDNA sequence were subcloned into the prokaryotic expressive vector pGEX-4T-l. The recombinant plasmid was identified by enzyme digestion and DNA sequencing. E. coli BL21 was transformed with the positive reconstructed expression vector and induced by 0.08mM IPTG for 4 hours at 25 C. After gene transfecting, the stable expression E. coli BL21 was obtained. Purified sTALL-1 was eluted from GSTrap affinity chromatography after thrombin cleavage. The...