Expression Of Three Centipede Toxin Polypeptides And Discovery Of A New Enzyme Cleavage Site For Enterokinase

Animal toxin polypeptide has a wide range of functions,and it is very closely related to the physiological and pathological activities of organisms.In the same time,it is an important resource bank for developing new drugs and antibiotics.Among them,disulfide-rich animal toxin polypeptides can specifically act and regulate voltage-gated ion channels to activate or block the conduction of electrical signals.In the meanwhile,voltage-gated potassium channels(Kv)are the main targets of various centipede toxin polypeptides.In recent years,various studies have shown that Kv1.3 channel is an effective target for the treatment of autoimmune diseases.For aiming at the heterologous expression,purification and physiological activity of animal toxin polypeptides,and laying a foundation for the in-depth study of the structure and function of animal toxins and the development of new peptide drugs,the following aspects of research are carried out In this paper:Firstly,expression,purification and activity of Otostigmus beroni toxin KTX-OB10.The toxin polypeptide StKTX23 is a potassium channel inhibitor with physiological activity.The toxin KTX-OB10 and the toxin polypeptide StKTX23 are highly homologous peptide molecules.The centipede toxin peptide KTX-OB10 was digested by program(EMBOSS software).The primers were designed at the sites of small enterokinase(EK)and tobacco protease(TEV),PCR amplification and purification of PCR products.The DNA fragment of KTX-OB10 was inserted into p GEX-4T-1 expression vector to construct the recombinant protein peptide p GEX-4T-1-KTX-OB10.The expression of fusion peptide was induced in prokaryotic bacteria E.coli Rosetta by induction expression technology.Glutathione(GST)tags were cleaved by different enzymes and purified by RP-HPLC.The MALDI-MS-TOF molecular weight mass spectrometry was consistent with the theoretical molecular weight,and the expression yield of the target polypeptide was 0.9 mg/L.The whole cell patch clamp technique was used to detect the physiological activity,and the electrophysiological activity showed that there was no obvious potassium channel inhibitory activity.Secondly,discovery and verification of new enterokinase restriction site.It should be pointed out that the recombinant peptide EK-KTX-OB10 was digested by EK enzyme site,and a new small enterokinase specific site(TY site)was found in MALDI-TOF-MS mass spectrometry,that is,the sequence "DIDTR".Specific sites are the structures that restriction enzymes can cut,which can conveniently cut and install the needed genes and play an important role in the expression process.For this reason,the researchers redesigned the primers of KTX-OB10,removed the original small enterokinase EK enzyme cleavage site,inserted the specific site "DTIDTR",and designed the polypeptide TY-OB10 to verify the single effect of the newly discovered specific site.At the same time,this site was added to the polypeptide OBD1 which has been difficult to cut in our research group.The expression,purification,separation and identification were carried out again to verify its high efficiency and successfully completed the verification.Thirdly,Expression,Purification and Electrophysiological Activity of Defensin OBD4.Defensins can play an important role in the body’s innate immune system and can specifically regulate voltage-gated ion channels.Centipede defensin OBD4 has three pairs of disulfide bonds and has the potential to treat autoimmune diseases.Based on the previous KTX-OB10 peptide,the target peptide was successfully obtained by TEV enzyme digestion.In this experiment,the target peptide was successfully obtained by using TEV enzyme digestion site when the defensin OBD4 primer was designed,and the expression yield was 0.3 mg/L.The target peptide was successfully obtained by mass spectrometry identification.In addition,the electrophysiological patch clamp technique was used to verify the physiological activity of defensin.The results showed that OBD4 had potassium channel inhibitory activity in Kv1.3channel,which laid a material foundation for the study of defensin and related diseases.