oa Cloning, Overexpression And Molecular Characterization Of Four Superantigens: Production Of Protein Therapeutics For Cancer Treatment

Background Bacterial superantigens (SAGs) are potent T cell stimulatory molecules and comprise a large family of disease-associated proteins. Superantigens bind to APCs on the outside of the MHC class II molecule and to T cells via the external face of the T cell receptor (TCR). This enables them to activate up to 20% of resting T cells, whilst conventional antigen presentation results in the activation of 0.001- 0.0001% of the T cell population. These biological properties of superantigens make them very attractive for use in immunotherapy. Objectives 1.Codon optimization, for maximum expression in E. coli and synthesis of four superantigens genes 2.Cloning, Overexpression, purification and molecular characterization of four superantigens Methods The four superantigens codon-optimized genes were produced by Geneart GmbH Company (Regensburg, Germany). Additionally, we introduced several new restriction enzyme sites into the gene, e.g. NdeI and HindIII sites were placed at the 5' and 3' ends of the gene, respectively to facilitate the cloning. Each gene was excised from pGA/sAg by digestion with NdeI and HindIII and inserted into similarly digested pET28a DNA using T4 DNA ligase. The newly formed construct of each gene was transformed into competent cells of E. coli BL21 (DE3) RIL. The expression of each superantigen was done by induction using IPTG. The induced cells were harvested by centrifugation and the total proteins were produced by sonication. The expression analysis was performed using SDS-PAGE. Novel SAGs (TSST, SPE, SEA and SEB) were tested in vitro for their superantigenicity and anticancer activity using peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers and prepared by Ficoll-Paque gradient centrifugation.Superantigenicity was assessed by monitoring the SAG-induced release of PBMCs cytokines. PBMC were treated in the presence or absence of SAGs for 24 h and the release of IL-1β, IL-8, IL-10, TNFα and IFNγ measured by Luminex assay. Furthermore, 3 different colon cancer cell lines were treated with SAGs in the presence or absence of PBMC for 48 h. Inhibition of cancer cell proliferation was measured by MTT assay. Results In this study, four synthetic genes coding for four different superantigens, SEA, SEB, TSST-1 and SPEA were codon-optimised and synthesized for maximum expression in E. coli using the vector pET28a. Our work showed that in all cases the recombinant superantigens were expressed to 40% of the total host protein. We managed to optimize the condition to obtain 20% of the recombinant superantigens in a soluble form. The purification of the soluble His-tagged recombinant superantigens have been achieved in a single step by Ni2+ charged column chromatography. Superantigenicity assay by measuring the released interferon gama showed that the four recombinant superantigens are active with superantigenicity functions. Conclusion The successful cloning and expression of four codon optimised synthetic superantigens genes will provide the recombinant protein which will be modified for production of safer superantigen for cancer immunotherapy.