Construction Of Expression Vectors And Functional Identification Of Butelase 1

Butelase 1 is a novel Asx?Asp/Asn?-specific peptide ligase can cleave the bond of peptide and protein as a protease.More importantly,it can efficient catalyze non-native linear peptide or protein from various organisms cyclization to form a head-to-tail cyclic backbone,which contribute to the stability against degradation by enzymes,chemicals and heat.In order to obtain soluble Butelase 1 protein,this research constructed four recombinant prokaryotic expression vectors and utilized fusion tags technology to expression soluble Butelase 1 protein in E.coli prokaryotic expression system.Subsequently,an eukaryotic expression vector of P.pastoris was constructed,and Butelase 1 protein was expressed by P.pastoris eukaryotic expression system.The enzyme activity of butelase 1 protein was detected by cleavage and cyclization reaction.The main experiments are as follows:1.Butelase 1 gene was inserted into p ET21 b prokaryotic expression vectors with fusion tags.The recombinant vectors were transformed into E.coli BL21?ED3?to induce the expression of fusion protein after bacteria PCR identification,enzyme digestion and sequencing identification.The prokaryotic expression of soluble fusion protein was purified through Ni-NTA affinity chromatography,and SDS-PAGE indicated that His₆-Grifin-Butelase 1 and His₆-GST-Butelase 1 fusion protein were expressed as inclusion body,His₆-Nus A-Butelase 1 fusion protein was mainly expressed in soluble body and some was in the inclusion body,His₆-MBP-Butelase 1 fusion protein was almost expressed in soluble body.Western blot identified that the His₆-Tag monoclonal antibody can specifically recognized the purified His₆-Nus A-Butelase 1 and His₆-MBP-Butelase 1fusion protein.2.Butelase 1 gene was ligated into the yeast expression vector p PIC9 K to construct P.pastoris eukaryotic expression vector p PIC9K-Butelase 1,the gene was integrated into the genome of P.pastoris GS115 by electroporation,secretion of target protein was induced by methanol.Approximately 400 ?L Butelase 1 protein was obtained after centrifugal,vacuum filtration and protein concentration from the expression production.Western blot analysis showed that there had soluble Butelase 1 protein expression.3.The His₆-Spy Tag-sf GFP-Spy Catcher?43.2 k D?protein,expressed by E.coli prokaryotic expression system,was cleaved by Butelase 1 protein expressed by P.pastoris.SDS-PAGE showed that the original protein was cleaved into two polypeptides,one peptide fragment was about 16.4 k D?His₆-Spy Tag-Spy Catcher?and another peptide was about 26.8 k D?sf GFP?.Indicating that Butelase 1 protein expressed by the P.pastoris eukaryotic expression system has proteases activity.Utlized Butelase 1 protein to catalyze linear peptide G-LL37-NHV?4900 Da?.The reaction products were identified by Tricine-SDS-PAGE,showed that there were two bands,one was the cyclized peptide G-LL37?4646 Da?and the other one was the original linear polypeptide G-LL37-NHV,this experiment demonstrates that Butelase 1 protein has cyclase activity.In conclusion,we constructed four recombinant prokaryotic expression vectors with different fusion tags: p ET21b-His₆-Grifin-Butelase 1,p ET21b-His₆-GST-Butelase 1,p ET21b-His₆-Nus A-Butelase 1 and p ET21b-His₆-MBP-Butelase 1.Screened MBP tag was the best fusion tag,and Nus A tag was the next-best tag can remarkably promote Butelase protein soluble expression.In addition,we also constructed one P.pastoris yeast expression vector,used P.pastoris GS115 host bacteria to induce target protein secretion expression,obtained eukaryotic expression of soluble butelase 1 protein,which has protease and ligase activity.This study showed that soluble Butelase 1 protein can express by E.coli prokaryotic expression system and P.pastoris eukaryotic expression system.And Butelase 1 protein expression by P.pastoris expression system can catalyze linear peptide cyclization,which provided experimental technical preparation for the production of cyclic antimicrobial peptide and would be benefit for the design and development of new cyclic peptides.