Intein Based Protein Splicing And Its Application

Inteins are internal protein sequences capable of catalyzing a protein splicing reaction by self-excising from a precursor protein and simultaneously joining the flanking sequences with a peptide bond.According to the structural characteristics,inteins can be divided into three categories,canonical inteins,mini inteins and split inteins.The protein splicing mediated by canonical inteins and mini inteins are referred to cis-splicing.Split inteins have separate pieces(IN,N-intein and IC,C-intein)locating in two different open reading frame that associate noncovalently to initiate a protein trans-splicing reaction joining two polypeptides.Inteins have become increasingly useful tools in protein research,protein engineering,and some other fields.For example,inteins have been used in synthesis of toxin protein,the activity control of target protein,gene therapy,protein modification,labeling,protein cyclization,protein purification and so on.In this thesis,we studied the Ssp DnaX mediated protein splicing when flanking Proline;The Ssp DnaX splicing in KSCDKTH amino acid sequence;Improving the splicing activity of Ter ThyX through directed evolution;A comparative study of natural Cne PRP8-S0 and improved Cne PRP8-E-S0;The application of protein purification based on two purification tags;The application of intein mediated RicinA splicing.The application of polyester fabric treatment by intein trans-splicing.Research content in the paper including following seven aspects:1.In pMST system,the residues at +2 or both at-1 and +2 positions were mutated to proline to test the intein splicing activity,which were Ssp DnaX,Ter DnaE-3 and Npu DnaE mini intein with high splicing activity.We found that Ssp DnaX mini intein had 100% splicing activity.Then we splited the Ssp DnaX mini intein into three split inteins(S0/S1/S11),and tested the splicing activity in vivo and in vitro.Research results show that Ssp DnaX-S1/S11 had high splicing activity when proline was at the +2 position or at both-1 and +2 positions both in vivo and in vitro.It was found for the first time,Ssp DnaX mini intein and S1/S11 split intein show high splicing activity when flanking proline residue.This finding contributes significantly to the toolbox of intein-based technologies by allowing the use of inteins in proteins having a proline at the splicing point.And it also provides information for the 3-D structure research of Ssp DnaX intein.2.The extein sequence of intein inserting site was mutated into KSCDKTH and the splicing activity of Ssp DnaX-S0/S1/S11 and Ter ThyX-S0/S1/S11 split inteins were tested in pMST system.Research results show that,Ssp DnaX-S0/S1/S11 show high splicing activity(~100%)both in vivo and in vitro.This finding contributes significantly to the toolbox of intein-based technologies by allowing the use of inteins in proteins having KSCDKTH sequence at the splicing point and provides a basis for the feasibility of the synthesis of antibodies.3.In pKH system(kanamycin resistant system),the splicing activity of Ter ThyX mini intein was improved to ~100%.The directed evolution can improve the intein splicing activity through simple random mutation,which is good for intein application in protein engineering.The intein obtained from directed evolution has higher versatility,it can be used to splice protein with different extein sequences.Moreover the mutation amino acids of intein can be obtained by DNA sequencing to research the relation of intein structure and splicing activtity.4.In pMST system,a comparative study was performed of the splicing rate,the splicing kinetics,and the N-cleavage and C-cleavage efficiency of Cne PRP8-S0 and Cne PRP8-E-S0.Research results show that the Cne PRP8-E-S0 show higher splicing rate and splicing efficiency than Cne PRP8-S0,especially with +1 position Ser mutated to Cys.The N-cleavage of Cne PRP8-S0 and Cne PRP8-E-S0 had little difference.For C-cleavage,the Cne PRP8-E-S0 had an advantage over Cne PRP8-S0.Here,by comparing the natural Cne PRP8-S0 and improved Cne PRP8-E-S0,we found that the inteins with functional advantages can be obtained by directed evolution,so that the inteins can be application better and more widely.The Cne PRP8-E-S0 can be used not only in splicing different target proteins,but also protein purification due to its C-cleavage efficiency and rate.Moreover,the Cne PRP8-E-S0 is more advantage than natural split intein Npu DnaE,showing high splicing activity with non-native +1 amino acid.5.Here we present a dual purification system.The system utilizes SUMO in series with hexahistidine(His6)and a novel Ssp GyrB S11 split intein in conjunction with a chitin-binding domain(CBD)as affinity tags.The structure of dual purification system(His6-SUMO-TP-Ssp GyrB S11N-CBD)was that,His6-SUMO tag was fused to the N-terminus of target protein,Ssp GyrB S11N-CBD was fused to the C-terminus of target protein,the target protein was in middle.Research results show that the MBP and Trx protein both have high purity(MBP,>95%,2.5-5mg/L)(Trx,>95%,4-8 mg/L),and the ESI-MS molecular weight result of Trx show the protein obtained form dual purification system has uniform N-and C-terminus.The system solved several purification problems,obtaining protein with high purity and uniform terminus,make it widely used for the protein applied into the protein research.The protein obtained from dual purification system can be used in proteomics research,and the dual purification system make it rising to a new level of protein purification method,resolving the protein purity and ununiform terminus once.6.Intein can be used to produce Ricin toxic protein.This research had two parts,the first part,the RicinA(mutate the amino acid of RicinA)was reformed to provide basis for intein high splicing activity.The toxicity of RicinA after mutation was detected by a curve of RicinA and toxicity.The second part,different insert sites of RicinA(site1 and site 2)were chosen to test the splicing activity of Cne PRP8E-S0 split intein.The curve of RicinA and toxicity were obtained.In addition,we found that the three mutations did not affect the toxicity of RicinA and Cne PRP8E-S0 spliced slightly in site2 with ~31% splicing efficiency.These findings provides a basis for the splicing of RicinA in vitro.It is also proved that the feasibility of intein splicing RicinA to achieve the specific killing of tumor cells.7.Polyester fabric finished by MT fusion protein.Due to its excellent properties of durability and easy washing,polyester fabric are widely used in clothing and other fields.However,polyester fabric has the disadvantages of poor hydrophilicity and poor antistatic property because of the lack of hydrophilic groups in its molecular structure.MT fusion protein using for polyester fabric finishing was obtained by intein trans-splicing,combining the MBP and Trx protein.The hydrophilic groups of MT fusion protein exposed on the surface of the fabric,enhanced the hydrophilic property of the fabric,decreased the half-life,and improved the antistatic property.This paper lays a theoretical foundation for studying the protein finishing of PET fabric and further development of the new functional polyester fabric with health care.