Construction And Use Of N-Terminal Cysteine-Free Split Inteins For Protein N-Terminal Labeling

Protein is the main executor of cell function. In many cases, the realization of a cell function is the result of multiple protein interactions. In recent years, with the development of protein labeling, the study of proteins has been into a rapid development stage. The research maninly include two directions:"in vivo" and "in vitro". In vitro, the studies mainly focused on purified proteins, placing them in a controlled environment, in order to obtain functional information; in vivo, the experiments focused on the proteins'functional role in the cell or the entire organization and thus we can understand the active central and corresponding regulary mechanisms.The protein engineering and protein modification in vivo or vitro could help to explore the proteins'biological and structure function. There are variety of chemical methods and the use of intein'trans-splicing for protein labeling. The former one is relatively sample, but there are many shortcomings, such as the specificity is not strong, chemical labels easily interfere with the activity of labeled proteins, need higher protein concentration, etc. Protein trans-splicing, which is catalyzed by split-intein, provided another avenue for protein labeling. Although it overcomes the disadvantages of the chemical labeling, it still has some urgent problem:the fusion protein is instability, poor solubility, low activity and poor general. In this paper, on the basis of the protein trans-splicing, we selected some inteins with fewer cysteines, and constructed the inteins into split inteins with higher splicing activity and N-terminal cysteine-free or cysteine-free by site directed mutation and overlap PCR. Then the inteins'splicing activity was detected by SDS-PAGE and Western Blot. For the active intein, we detected its application potential as labeling method in our laboratory'TM system. Finally, we selected the target protein for protein N-terminal specific labeling.The topic selected TerNdse-2, TX-S1, HaVol Pol, Msm DnaB-1, Arsp FB24 and PP-PhiEL ORF40 for the study. Msm DnaB and Arsp FB24 are Class III intein, and the rest are standard intein. The first amino of TerNdse, TX-S1 (Cl/S, HaVol Pol, Msm DnaB-1å'ŒArsp FB24 are serine, serine, serine, alanine and glycine, respectively. There are also very few cysteines in the internal of these intein, it is very useful for subsequent site-directed mutation and protein N-terminal labeling.First, we detected these inteins'original splicing activity in vivo, pMTerNdse-2 has very low splicing activity, pMTX-S1 (Cl/S) has high splicing activity, pMHP' splicing efficiency is 100%, pMMD (the first cysteine is free) has weak activity and pMAF has no splicing activity.Then, we changed the cysteines in the intein to serine by overlap PCR, and restored the first cysteine in pMMD. pMTerNdse-2,pMTX-S 1 (Cl/S) and pMHP mutation in cysteine completely without any splicing activity, while pMMD has high splicing activity.For pMHP, four internal cysteines were mutation into serine, respectively. We found that the second cysteine plays a decisive role for its splicing. Then the second cysteine was replaced by threonine and glycine, which have similar functional groups. Fortunately, we got two cysteine-free mutant of HP, pMHPG and pMHPT, they both have high splicing activity, and is very important for abtain a universal labeling means.We constructed the S1,S0 and S11 split intein in vivo of pMTerNdse-2, pMMD, pMHP, pMHPG and pMHPT and detected their splicing activity by Western Blot. pMTerNdse-2, pMHP and pMHPG'split intein has no activity in vivo, pMHPT'S1 split intein has high activity, pMMD-SO'activity is 100%.After modeling and alignment of SWISS MODLE and Protean, we re-selected the other fracture sites. pMHP, pMHPG and pMHPT are ten new fracture sites respectively, pMMD is five new sites, constructed their clones and detected their activity by Western Blot. The results showed that, pMHP-F2 and F8 have higher splicing activity, pMHPT only F2 has some splicing activity and pMMD-F4 has very high activity.Expression plasmids were constructed of pMHP-F2, pMHP-F8,pMHPT-S1, pMHPT-F2,pMMD-S0,pMMD-F4 and pMTX-S1 (Cl/S) for 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 Amylases 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 pMTX-S1 (Cl/S) and pMMD-SO split-inteins had splicing activities which were assessed through Western blotting.The intein in vitro has splicing activity, we choose TXS1 for protein N-terminal labeling by TM system. The TX-S1N was built into the expression vector PEDHC containing the SUMO protein gene sequence, then purified the TX-S1N and TX-S1C by His-tag in vitro. The purified TX-S1N and thiol dye were mix together in the molar ratio of 1:5 for four hours. The unreacted dye needed dialysised through dialyasis bags. And then the SUMO was removed by SUMO enzymes for 1 hour. The L-TX-S1 and TX-S1C were reacted for three hours in Splicing Buffer, and detected by SDS-PAGE, the results of the fluorescence response can be observed by 346-442 nm UV.The issue has achieved protein N-terminal labeling, it is not only a complement of previous studies, but also overcome the disadvantages of chemical synthesis and protein labeling. At the same time, we found a very interesting phenomenon:in pMHP and pMMD intein, a cysteine in their non-conserved region was able to determine the activity of the intein, and there is no literature report about this. Its chemical and biological mechanism worth continuing to study, and believed it to be a good complement for the theory of intein splicing. In addition, we got two cysteine-free intein mutantion, although they did not get a break in vitro splicing, we will continue to study, hoping to abtain multiple split intein with high splicing activity. In which it not only provides a tool for protein N-Terminal labeling, but also provides a general intein and a new way of labeling for both C-terminal labeling and two-terminal labeling.