Peptide Cyclization Through Trans-splicing Of Split-inteins

Cyclopeptides are a kind of cyclic compounds with stable structure and wide biological activity,which have wide application.In particular,the circular antimicrobial peptide has the characteristics of heat stability,broad antibacterial spectrum,small molecular weight,low immunogenicity,etc.Because of its unique antibacterial mechanism,it is not easy to produce tolerance,which is expected to develop into a new generation of peptide antibiotics.It is very important to design,modify and prepare natural cyclopeptides which will be helpful to further study their mechanism and improve their antibacterial activity.Chemical synthesis is a common method for the preparation of cyclopeptides,which has some limitations,such as high cost,easy to form polymer by-products and cumbersome cyclization steps.In this study,we used split intein mediated protein trans-splicing to prepare the cyclic antibacterial peptide.Split inteins which derived from canonical inteins,can self-catalyze and mediate protein trans-splicing to form mature protein.The reaction is enzymological and can occur under physiological conditions with low molecular concentration.The splicing domain of the break protein intron is divided into N-terminal splicing domain(I_N)and C-terminal splicing domain(I_C),which can be located in different open reading frames.In protein trans-splicing,N-terminal splicing domain and C-terminal splicing domain recognize each other through non-covalent interactions.The splicing active center is reconstructed,and then self-catalyzed from the precursor protein,while the flanking exteins are connected with new peptide bonds.Therefore,in the process of peptide cyclization,the target peptide is inserted between the splicing domains of the two ends of the split intein,and the end-to-end peptide is cyclized by trans-splicing.In this study,four kinds of bacteriocins(AS-48,garvicin ML,butyrivibriocin AR10 and uberolysin)were selected.They are natural cyclic peptides with strong antibacterial activity produced by different bacteria.At the same time,CPE and RB,two novel split inteins with high splicing activity and good generality were selected.Firstly,two universal vectors were cloned and constructed by PCR and overlapping extension PCR.The C-terminal splicing domain and N-terminal splicing domain of the two kinds of split inteins were inserted with the tag sequence flag as the linker,respectively.Then through seamless cloning(without introducing any extra amino acids),the flag sequence in the universal vector was replaced by four kinds of bacteriocins.The bacteriocins were inserted between the splicing domains at both ends,and eight recombinant expression plasmids were constructed.The results showed that only two bacteriocins,butyrivibriocin ar10 and uberolysin,were successfully expressed in E.coli.It was found that only the bacteriocin uberolysin in the recombinant protein was successfully spliced by RB,but the splicing rate in vivo was relatively low.Because of the lower molecular weight of cyclopeptide it was difficult to detect and purify.In the follow-up experiments,we used denatured purification and non denatured purification for the recombinant protein under different temperature induction.Yet the trans-splicing of the split intein in vitro was not detectable.In order to further verify the cyclization of uberolysin,we modified the recombinant protein later,introduced the small protein tag sequence c-myc,and verified the cyclopeptide uberolysin by SDS-PAGE,tris Tricine SDS-PAGE and Western blot.The preliminary results showed that the splicing and cyclization of bacteriocin uberolysin in E.coli.In the future,we will further identify the existence of cyclopeptides by HPLC and mass spectrometry.This method provides a new way for the preparation of bacteriocins,and also lays a foundation for the further application of circular antimicrobial peptides.