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Functional overexpression and purification of a codon optimized synthetic four superantigens in Escherichia coli: Production of superantigens with improved safety and efficacy for cancer immunotherapy (LB582)
Author(s) -
Bashraheel Sara,
AlQahtani Alanod,
AlSulaiti Haya,
Althani Amna,
Goda Sayed
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb582
Subject(s) - superantigen , t cell receptor , recombinant dna , antigen , biology , microbiology and biotechnology , t cell , streptococcus pyogenes , antigen presentation , immunology , immune system , staphylococcus aureus , bacteria , gene , biochemistry , genetics
Bacterial superantigens (SAGs) are potent T cell stimulatory molecules and comprise a large family of disease‐associated proteins. The best‐characterized members of the superantigen family are staphylococcal enterotoxins (SEs), which are secreted by Staphylococcus aureus , and streptococcal pyrogenic exotoxins (SPEs), which are secreted by Streptococcus pyogenes Unlike conventional antigens they are not processed internally by antigen presenting cells (APC), and are thus not displayed as peptide antigen in the peptide‐binding groove of the MHC class II molecule. 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. 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 indicated that in all cases the recombinant superantigens were expressed to 40% of the total host protein. We managed to optimise 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 Ni 2+ charged column chromatography. The superantigenicity of each synthetic recombinant SEA, SEB, TSST‐1 and SPEA was determined by 2 methods. First method includes 3H‐thymidine incorporation assay as a measure of T‐cell proliferation i.e. T‐cell stimulation by modified SAGs. The second part of the superantigenicity assays includes measurement of cytokines produced by peripheral blood cells. The successful cloning and expression of four codon optimised synthetic superantigens genes will provide the recombinant protein for production of safe superantigen for cancer immunotherapy. Grant Funding Source : Anti‐doping lab‐Qatar