Preparation Of A Novel Affinity Purification System Mediated By Split Intein Npu DnaE

Npu DnaE intein is a naturally split intein with higher rapid trans-splicing and cleavage efficiency than most inteins. The N-terminal segment（I_N） and C-terminal segment（I_C） of split intein have strong ability of binding and mutual recognition, and its splicing ability can not be activated before the combination of I_N and I_C. So, split intein-mediated system provides a better choice for protein purification and avoids premature cleavage during protein expression. In this paper, based on split intein Npu DnaE, we built several affinity ligand N and fusion protein expressing segments CG with different structures. Under different conditions during purification, we hope to find out a group of N and CG which fit best with purification process, and build a new purification system with them. The main works were carried out as follows:1) Constructing affinity ligands N and fusion protein expressing segments CG in Npu DnaE mediated protein purification system. Two mutation sites C1 A and D118 G were introduced in Npu DnaE to elevate cleavage efficiency, and a Cys was added to the C-terminal of I_N for the purpose of connecting I_N with Purose 6 Fast Flow by the –SH of Cys. Affinity ligand N and fusion protein expressing segment CG with different structures were built by adding His-tag of different length to N- or C-terminal of N and CG segments. After expression and purification, we successfully get affinity ligands N1, N2, N₃ and fusion protein expressing segments CG1, CG2.2) Comparison of the cleavage efficiency between different groups of N and CG under media-independent condition. The results of DTT induced cleavages showed that adding peptide on N-terminal of both I_N and I_C segments could lower the cleavage activity due to steric hindrance of the interaction between I_N and I_C. By comparision, we chose four groups with higher activity--- N2&CG1, N2&CG2, N₃&CG1 and N₃&CG2, and their cleavage time were 10 h, 4 h, 1 h, 1 min, seperately. In the process of studying the inhabitation effect of Zn²⁺, we found Cys1, except His48, Asp118 and +1Cys, might be another residue important for zinc inhabitation. The inhabitation effect was activated only if the C1 A mutation was reverted to Cys1 in all the four groups. Considering the specific condition in chromatograpghy process, we chose N₃&CG1 and N₃&CG2（with Cys being the first residue in I_N） as feasible schemes for building affinity purification system.3) Construction of Npu DnaE mediated purification systems and evaluation of their purification performance. By coupling affinity ligand N₃ with sepharose 6FF, we prepared affinity chromatography medium N₃ Purose 6 Fast Flow and built purification system N₃ Purose 6 Fast Flow&CG1 and N₃ Purose 6 Fast Flow&CG2. The purity of target protein GFP deriving from both of the two systems was much higher than which from Ni-affinity purification system. The analysis results showed that N₃ Purose 6 Fast Flow&CG1 had higher purification efficiency（54.7%） but longer holding time（30 min） compared with N₃ Purose 6 Fast Flow&CG2（50.9% and 2 min seperately）. Finally, we confirmed that both two systems were the feasible intein-mediated purification system.