Heterologous Expression,Purification And Activity Of Animals Polypeptide Toxins

Animal toxins have a wide range of functions,and play different roles in the blood system,nervous system,immune system and bacteria and fungi,constituting the important resource base for the development of new drugs and clinical treatment tools.Disulfide-rich peptides constitute the most toxic polypeptide richness and diversity.Polypeptide toxins which contain 2-4 of disulfides can specifically target and adjust the voltage gated ion channels,which results in a blocking or inhibiting the transmission of cellular electrical signals.Thus it has extremely important research value.In view of the heterologous expression and purification of animal toxins,this paper has carried out the following two works,which lays a foundation for further study of the structure and function of toxic toxins and the development of new peptide drugs.1?Expression,purification and activity analysis of?-SLPTX-Ssm1b.?-SLPTX-Ssm1b is certified from S colopendra subspinipes mutilans cDNA library,which is homologous with the ?-SLPTX-Ssm1a,an inhibitor of voltage sensitive potassium channels.Both of them have three pairs of disulfide bonds with the same homology as 39%,which leads to a result that they might have similar spatial structures and similar physiological activities.In this study,by using the expression vector of pET-43-His-SUMO,we transfer it into SHuffle^TMstrain and then use the automatically induced expression to obtain recombinant protein.Then it is purified by nickel column and RP-HPLC,and incised by Ulp1 kinase.Finally it is identified by MALDI-TOF mass spectrometry.Its molecular weight is 5532.1870 Da,which is consistent with the theory value 5532.3 Da.Its yield was 1 mg/L.But the electrophysiological activity showed it had no significant effect on rKv1.3 and rKv1.7 in rats.2?Expression and purification of U1-PONTX-Ae1d/e/g.A total of four toxin sequences?U1-PONTX-Ae1d/e/f/g?were isolated and identified in Anochetus venom,which are highly homologous with U1-PONTX-Ae1a,an inhibitor of voltage sensitive calcium channels.Both of them have two pairs of disulfide bonds with U1-PONTX-Ae1a,which results in the fact that they may have similar activity with U1-PONTX-Ae1a.In this study,U1-PONTX-Ae1d/e/f/g was expressed in E.coli.We successfully obtained U1-PONTX-Ae1d/e/g,and their production is about 0.3 mg/L.And it laid the foundation for further activity testing and drug screening.