Development Of Chemical Lysis Method For Recombinant E.coli And Its Influence On Protein Purification

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a surface glycoprotein involved in cytotoxic T cell activation. Only activated T cells express CTLA-4 on their surface, which can compete with CD28 for binding the same B7 ligand to down-regulate T cell activation. The whole molecule of CTLA-4 consists of an extra-cellular domain, transmembrane region and a cytoplasmic tail. It has been shown that the extracellualr domain of mammalian CTLA-4 can be used to target the fused antigens to antigen presenting cells and thus can serve as a new-type molecular adjuvant.We use recombinant engineering bacteria Escherichia coli M15/pQE-31-CTLA4- VP2S,CTLA4-VP2S expressed as inclusion bodies in E. coli after IPTG induction is a target protein, the molecular weight of which was 42KD, to study the optimum lysis solution, recombinant engineering bacteria was chemical cleaved by 5 different solutions containing Jiamei washing powder. On the base of above studies, we processed the pilot study of the optimum chemical lysis conditions. The optimum conditions were determined as follows:solutionⅢ(0.1%Triton X-100, 100mM NaCl, 25mM Tris-HC1(PH8.0) )with 1.5% jiamei washing powder, temperature 4℃,15min. we processed the pilot study of the chemical lysis effect of different strains with the same plasmid and the same strain with different plasmids. The results showed that the effect is different with different strains but without plasmid. To study of the chemical lysis effect for expressed protein, pGEX-VP72S(M15),pQE-31-CTLA4-VP2S(M15)are induced by IPTG and cleaved.Affinity chromatography has been set up to purify the recombinant protein. The results showed that the purity was 93%, and the final protein recovery of soluble protein GST-VP72S was 42.6%. Inclusion body CTLA4-VP2S could be obtained due to lysis, inclusion body washing, The recombinant protein was then purified by Protino Ni-TED2000 packed columns Affinity chromatography. The purity was 90%, and the final protein recovery was 37.8% .