Kinetic Analysis Of Trans-splicing Of Novel Split-inteins In Vitro

Intein is an internal protein sequence in host protein precursor. It can autocatalytically self-excise and concomitantly splice together the flanking sequences (N-and C-exteins) with a peptide bond to produce the mature host protein. This process is termed as protein cis-splicing. Split-intein is an intein which splits in specific internal site and two parts of the intein (N-intein and C-intein) encoded by two separate open reading frames located apart in the genome, respectively. In the process of splicing, the N-intein and C-intein rapidly self-associate and reconstitute protein trans-splicing activity.Novel split-intein is artificially reconstructed whose splitting site is close to the N-terminal or C-terminal of intein. Protein trans-splicing mediated by split-intein has made some achievements in protein purification, gene therapy, cyclic peptides preparation and the production of macromolecular complex proteins. Novel split-intein significantly expands the utility of split-inteins, providing the potential to add any number of desirable chemical groups onto the N-teriminus or C-teriminus of target proteins. We need to comprehensively understand enzymatic kinetics of novel split-intein before apply them, therefore, it is important to study kinetics of novel split-inteins.Novel split-inteins SX-S1, TX-S1, SX-S11 and TE3-S11 were selected for kinetics studies in this work. The N-intein of the S1 split-inteins is only 11-aa in length, and the C-intein of the S11 split-inteins is only 6-aa in length, thus they can easily be chemically produced to be added biotin or fluorescent labels to the N-terminus or C-terminus of recombinant proteins or drugs.Expression plasmids were constructed which included the sequences of N-precursor protein or C-precursor protein. N-precursor protein contains maltose binding protein and N-intein, and C-precursor protein contains C-intein and thioredoxin. Because maltose binding protein tag in N-precursor protein and 6×His tag in C-precursor protein can specifically combine with Amylose resin and Ni-NTA resin respectively, N-precursor protein and C-precursor protein were purified for reaction in vitro by the method of affinity chromatography.The purified N-precursor protein and C-precursor protein were mixed together in the molar ratio of 1:1. The results demonstrated that SX-S1, TX-S1, SX-S11 and TE3-S11 split-inteins had splicing activities which were assessed through Western blotting. Different N-precursor protein and C-precursor protein were recombined to have a cross tran-splicing reaction in vitro and detected the splicing activities. No combinations had cross reaction activities except TX-S1N/SX-S1C. It is helpful for using many different split-inteins to label multi-segments of a target protein or multiple interest proteins. Then the optimal conditions for split-inteins trans-splicing were explored, including studying the influence of DTT concentration, temperature, NaCl concentration and pH value on the efficiency of splicing. When there was no DTT in the reaction system, the splicing efficiency decreased significantly, even to zero. However, the efficiency of splicing was comparably stable under other conditions.The purified N-precursor protein and C-precursor protein were mixed together in the molar ratio of 10:1 to study the kinetic of these novel split-inteins. The results from kinetic analysis demonstrated that the trans-splicing efficiency of split-inteins SX-S1, TX-S1, SX-S11 and TE3-S11 reached-96%,-50%,-93% and -87%, and the rate constants were determined to be (2.68±0.39)×10-4 s-1, (2.19±0.33)×10-3 s-1, (3.94±0.91)×10-4 s-1 and (3.43±0.59)×10-4 s-1,respectively.The potential application of split-inteins for site-specific protein labeling results in that the protein labeling is no longer confined to traditional amino acid-specific labeling. Understanding the mechanism of split-intein splicing pathway has led to the development of many applications in the fields of protein engineering, development of protein drugs and research of protein structure and function.