IDENTIFICATION, CLONING AND RECOMBINANT EXPRESSION OF IMMUNE MODULATORY TIR PROTEIN FROM PROBIOTIC Bifidobacterium breve
Project no. 763 · Status: Kapanmış · 2016
Summary
Studies on probiotics which have immunomodulatory properties, show that probiotics produce molecules which can affect the immune system. In this interaction, Toll like receptors (TLR) on cell surface are also thought to play a role. Several bacterial TIR domain proteins structurally mimic the human adaptor TIR domain proteins and they manipulate the immune response by altering the signal transduction. Following these, the presence of probiotic TIR domain proteins was searched using bioinformatics tools. A putative TIR domain sequence in the genome of Bifidobacterium breve was found. In this thesis, cloning, recombinant production and purification of putative TIR domain protein from B. breve (BbTIR) was investigated. It has been confirmed by bioinformatics methods that BbTIR contains conserved TIR domain patterns by multiple sequence alignments. bbtir gene was cloned into pETM11-SUMO3GFP and pET151/D-TOPO vectors. Protein expression from different cells and induction conditions were tested for optimum recombinant protein production. 6his-BbTIRV1 and 6his-BbTIRV2 protein was achieved from Rosetta 2 cells at 20 §C with overnight and 25 §C for 6 hours with 1 mM IPTG induction respectively. The recombinant proteins were purified by nickel affinity chromatography together with a protein which is thought to be a cleavage product and is equal to the moleculer weight of the TIR domain. The oligomeric structure was examined by gel filtration chromatography and protein was obtained in monomeric form in 50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 10 mM DTT buffer. Experimental results were supported by alignments and protein structure models from bioinformatics analyzes verifying the presence of probiotic TIR domain proteins with TIR domain structures.