Preparation Of Multiple Disulfide Boned-containing Peptide

There are many kinds of peptides rich in disulfide bonds,which have various biological functions and are a treasure house of natural resources.Compared with linear peptide,disulfide peptide has strong specificity and good stability,and has good potential to be developed into drugs.Correct disulfide bond pairing is the premise of its normal biological function.Although some conotoxin,scorpion toxin and spider toxin rich in disulfide bond have been successfully expressed by genetic engineering,it is still difficult to express and purify these peptide toxins.When selecting the expression and purification system,it is necessary to fully consider the properties of the expressed polypeptide,the expression level,the experimental cost,the expression cycle,the safety and the influence of the expressed polypeptide toxin on the expression system itself.Because the molecular weight of polypeptide toxin is very small and rich in disulfide bonds,it is sometimes very difficult to express it in the form of non fusion.Generally,the fusion expression is not only conducive to the detection and purification of expressed polypeptide,but also to enhance its solubility and stability.Combined with the properties of polypeptide toxins,it is suggested that the fusion of Trx,MBP and His tags,as well as the fusion of molecular chaperones,such as small ubiquitin like modified protein(sumo),can not only improve the expression and solubility of polypeptides,but also promote the correct folding and anti protease hydrolysis of polypeptides.With the continuous development of molecular biology technology and in-depth study of polypeptide toxins,it is expected that new suitable expression and purification systems for polypeptide toxins will continue to appear,and the development of gene expression and purification of polypeptide toxins will play an increasingly important role in the study of structure and function of polypeptide toxins.In this paper,four conotoxins CnⅢC,GⅢB,KⅢA and PⅢA containing three disulfide bonds were expressed in E.coli pET32a/pETDuet.Primers were designed according to the gene sequence,and conotoxins GⅢB and PⅢA were constructed into pET32a expression vector by double enzyme digestion.Conotoxins CnⅢC and KⅢA were constructed into pETDuet-1 expression vector.Then the conotoxin PⅢA was constructed into pETDuet-sumo expression vector by RF cloning.The recombinant plasmid was transformed into E.coli BL21(DE3)and induced by IPTG.The four conotoxins were highly expressed in the form of supernatant.For GⅢB and PⅢA,the purified peptides were obtained by twice Ni column purification,enzyme digestion and ultrafiltration tube purification.For CnⅢC and KⅢA,two small peptides were purified by nickel column,purified by MBP column and purified by gel chromatography Superdex 75.The molecular weight of the purified peptide was consistent with the theoretical molecular weight by mass spectrometry.Electrophysiological experiments showed that it had certain activity.In conclusion,we have successfully obtained four conotoxins CnⅢC,GⅢB,KⅢA and PⅢA with correct structure and activity in E.coli,which not only provides reference value for the preparation of other peptides rich in disulfide bonds,but also lays a foundation for large-scale production of recombinant conotoxins with high activity,low cost and high safety by foreign expression system.The successful establishment of E.coli Expression System of disulfide rich polypeptides will greatly accelerate the basic and applied research of disulfide rich polypeptides.