Internal-Site-Specific Protein Labeling Through Trans-Splicing

Protein labeling is essential for the study of protein function and structure.Nowaday,with the continuous development and maturation of protein labeling technology,protein specific site labeling has been widely used in the field of biological science,especially in the study of protein function.In order to achieve site-specific modification and labeling of the target protein,many methods based on molecular biology and chemistry for protein labeling have been developed at present,but they all have their own disadvantages and most of them can only do protein terminal site labeling but cannot be site-specific markers.Protein trans-splicing technology provides a new way for protein site specific labeling.Protein trans-splicing is mediated by split intein.The split intein consists of two parts: the N-terminal()and the C-terminal()of the split intein,respectively,located in two open reading frames(ORF).After the protein has matured, and can reidentify,and then reform the catalytic center.The split inteins are cleaved and the exons are connected by peptide bonds.At this stage,protein terminal labeling has been achieved by using the split intein,but the site-specific labeling of the protein is still not achieved.In this paper,we have achieved the specific labeling of the internal sites of proteins.This text finally selected two kinds of split inteins TE3S11 and RBS1.Among them,the N-terminal of S1 split intein contains only 11 amino acids,and the C-terminal of the S11 split intein only contains 6 amino acids.They were used for the target protein of fluorescent labeling.The maltose-binding protein binds to the N-terminus of the S11 type split intein to form an N-terminal precursor protein.The C-terminal of the S11 split intein and the N-terminal of the S1 split intein was labeled with the FITC to form an intermediate precursor protein.The C-terminus of the S1 type split intein and thioredoxin(Trx)were fused to form a C-terminal precursor protein.Intermediate precursor protein was synthesis by chemical methods.The N-terminal precursor protein was purified by Amylose Resin.The C-terminal precursor protein was purified by Ni-NTA.The N-terminal precursor protein,C-terminal precursor protein and intermediate precursor protein were spliced at a molar ratio of 1: 1: 1 and finally detected by SDS-PAGE,Western Blot,fluorescence and mass spectrometry.The final results showed that this paper can be site-specific protein labeling.Compared with the existing technology,the technology has many advantages,such as simple,universal and truly achieved internal site-specific labeling in this paper.It provides a new ways for protein labeling.And it has great potential application in protein engineering.