Preparation And Specific Toxicity Of Recombinant Toxin DAB₃₈₉(Gly₄Ser)₂-αMSH

Diphtheria Toxin (DT) is a single chain exotoxin which molecular weight is 58KD.It secreted by corynebactenum diphtheriae carrying DT gene. Eukaryotic cells are very sensitive to DT, only one or two molecules of DT, the complete DT molecule is cleaved into two fragments A and B, A fragments is residue 193aa, B fragment is 342aa, the receptor binding domain of the fragment B will bind to DT receptor, and the transmembrane domain of the fragment B will translocation the fragment A into the cytoplasm of the sensitive cells, and the fragment A in the cytoplasm catalyses the transfer of the ADP-ribosyltransferase group of NAD+ to elongation factor-2 (EF-2) , as a consequence, EF-2 is inactivated, and leading to the protein synthesis inhibition and cell death. They have a binding region, a translation region and a toxin effect region. The part of B can with main cell in sensitive of is combined by translocation and can turn the part of A into the cytosol.a-MSH is a single chain polypeptide, and made of 13 amino acids residues, and is a nerve incretion hormone. a-MSH have many role in body such as low toxicity, strong inflammation et al. It can use as tumor target because small weight, and combine with MSHR in surface of tumor cell.We have constructed recombinant toxin gene in which the sequences for the binding domain of diphtheria toxin, the DAB₃₈₉ (Gly₄Ser)₂-αMSH gene fragment was amplified from pET28a/DAB₃₈₉(Gly₄Ser)₂-EGF by PCR. The PCR products were digested by Nco I and EcoR I, and then inserted into pUC18T, and digested by Nco Iand EcoR I, then inserted into plasmid vector pET28a( + )/Nco 1+ EcoR I, new plasmid pET28a/ DAB389(Gly4Ser)2-aMSH was obtained. We transformed recombinant plasmid into E.coli BL21 (X DE3) for attained protein expression. Under induction with IPTG the recombinant toxin was expressed a soluble protein and tested by SDS-PAGE and Gal scanned analysis, DAB389-aMSH and DAB389 (Gly4Ser)2-aMSH protein reached 24.46% of the total protein.The designed fusion protein DAB389 (Gly4Ser)2-ctMSH was analyzed and compared with DAB389-aMSH using DNASTAR in some molecular biology characteristics. It was predicted that the antigenic index and surface probability of DAB389 (Gly4Ser)2-aMSH were all lower/less than that of DAB389-aMSH. The isoelectric point of them was 5.9.The suitable culture medium is M9B2, and E.coli strain is E.coli BL21 (k DE3) for attained protein production. Under induction with 1.4 mmol/L IPTG for high protein expression of the recombinant protein was studied, after fermentation the expression reached at the top level 6 hours after inducted with 1.4mmol/L IPTG concentration. The protein expression of the recombinant protein in total protein was 28.64%. The recombinant protein weight reached 16.5g/L.Cells was collected by centrifugation and lysed by sonication. Protein was precipitated by 55% ammonium sulfate and then purified by Cu Chelating Sepharose Fast Flow and Superdex G-25 chromatogphy the purity of DAB389 (Gly4Ser)2-aMSH was about 90.53%,then use Xa factor digested and preparative electrophoresis. With the above purification, the purity of DAB389 (Gly4Ser)2-aMSH was about 93.26%.We detected the recombinant protein assay in ten cell, and cytotoxicity of DAB389(Gly4Ser)2-aMSH on SP2/0, HeLa, A549, A375 cell growth was analyzed with crystal violet staining. The IC50 was 3.138^g/mL, 2.736jig/mL, 7.243|xg/mL, and 4.672ng/mL respectively.