Expression,Purification And Function Assay Of BCM-Fc-APRIL Blocker

Members of the tumor necrosis factor(TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. APRIL(a proliferation-inducing ligand, TNFSF13a) and BAFF(B cell activation factor of the TNF family, TNFSF13b) are TNF ligand family members displaying typical features of type Ⅱ transmembrane proteins that were discovered by homology searches of the mucleic acid databases. Through interactions with a transmembrane activator and CAML interactor(TACI),BAFF-R or B-cell maturation antigen(BCMA), BAFF/APRIL play a critical role in the growth, differerntiation and development of B lymphocyte onset and/or maintenance, class switch of antibody, maintenance of germinal center and T cell co-stimulation.BAFF/APRIL system(BAFF, APRIL and their receptors) play crictial role in autoimmune disease and onset and maintenance of B lymphoid cancer. Clinical research find that high expression levels of BAFF and APRIL in systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and malignant tumors. Therefore, targeting the BAFF/APRIL system may protect against autoimmunity and lymphoid cancers through the inhibition of pathogenic B and T cell function. Antibody or deceptive receptor is effective way to cure the autoimmune diseases by block the APRIL/BAFF.Molecular simulation technology provides a new means to protein research. In theory, it can be solve the problems of the structure predicition and functional analysis and protein engineering. It play a critical role in protein structure prediction and modeling work.It achieve the perfect combination of biotechnology and computer technology. Three-dimensional structural model of deceptive receptor BCMA-Fc fusion protein was finished by molecular simulation. It was predicted BCMA-Fc has the ability to combine APRIL.Pichia pastoris expression system is easy to operate, fast growth, low mutritional requirements and high expression levels advantages. Pichia pastoris GS115is expression host of gene BCMA-Fc. The plasmid pPIC9K is expression vector. The experiments found the target protein is N-terminal heterogeneity by the commercial plasmid pPIC9K was transformed into pichia pastoris. This is due to Kex2and Ste113have different restriction site in α-factor signal peptide sequence processing. The refolded plasmid pPIC9K that target protein have the original, homogeneous N-terminus in the expression of pichia pastoris. Pichia expression system as a eukaryotic expression system has advantages of protein post-translational processing and modification, include protein glycosylation. Glucoprotein have different molecular weight caused by different lengths of sugar chain. According to the characteristics of pichia pastoris glucoprotein, BCMA-Fc gene sequences was finished point mutations to protein was expressed in pachia pastoris with a uniform molecular weight. Otherwise, expression of exogenous gene is relevant to gene condon selection. Codon-optimized gene sequence of target protein was finished by expression system host on the codon usage preference. So optimized gene sequence of the target protein expression in pichia pastoris improved significantly. In order to further study the biological activity of BCMA-Fc, we need to do pichia pastoris fermentation studies and optimization of purification conditions. Optimization of pichia pastoris fermentation condition, include the composition of the medium, induced conditions(temperature, pH). Finally,we achieve a high expression of the target protein in pichia pastoris by optimizaed fermentation conditions. IgGlFc fragment of BCMA-Fc fusion protein can be bind the ProteinA that is beneficial to purification. The extracellular activity experiments shows BCMA-Fc have the ability to bind the ligand APRIL. The experiments of cell activity illustrate BCMA-Fc can block the proliferation of APRIL on Raji B lymphoma.In order to compare BCMA-Fc in different expression system, we chose the E. coli expression system to express it. The main advantages of expression of the target gene in E. coli:the host genetic background is clear and easy to control gene expression;easy to cultivate to express target protein. someone new E. coli host cells containing the thioredoxin and glutathione reductase gene is beneficial to correctly folded of protein contain disulfide bond, and enhance it’s the solubility.The pET23d plasmid was selected as expression vector to meet the host cell. The pET23d-BCMA-Fc was constructed and transformed to E. coli. The BCMA-Fc is express in the supernatant after IPTG induction. The purification was finished by rProteinA affinity, gel chromatography and Q sepharose. The extracellular activity experiments shows BCMA-Fc have the ability to bind the ligand APRIL. The experiments of cell activity illustrate BCMA-Fc can block the proliferation of APRIL on Raji B lymphoma and have no difference with BCMA-Fc was expressed in pichia pastoris.To further study the activity of the target protein, we have established the MOG antigen-induced autoimmune encephalomyelitis model.The experimental autoimmune encephalomyelitis is one of classic animal model of multiple sclerosis. It is the immune inflammatory demyelinating model of the white matter of the central nervous system by isotype, dissimilar nerve tissue antigen-induced. The pathological features is similar to MS. It shows CNS white matter demyelination and inflammatory cell infiltration. The analysis of the animal models of disease pathology section shows the EAE mice brain tissue and spinal cord have a large mumber of inflammatory cell infiltration in HE staining under light microscope. It is the foundation to target protein biological activity in vivo experimental studies.BCMA-Fc was expressed in the pichia pastoris and E. coli system and cell activity analysis shows target protein have complete biological activity. The MOG antigen-induced autoimmune encephalomyelitis model have the foundation to target protein biological activity in vivo experimental studies.