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Construction and Use of N-Terminal Cysteine-Free Split Inteins for Protein N-Terminal Labeling

Author: LiJia
Tutor: MengQing；LiuXiangQin
School: Donghua University
Course: Biochemistry and Molecular Biology
Keywords: Protein N-terminal tag N-terminal cysteine ??intein Cysteine ??intein Protein trans- splicing TM system
CLC: TQ463.6
Type: Master's thesis
Year: 2012
Downloads: 11
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Abstract


Protein is the main executor of cell function. In many cases, the function of the realization of a cell is the result of the interaction of a plurality of protein molecules. In recent years, with the protein labeling technology continues to evolve and mature protein has entered a stage of rapid development. Their study \Vitro studies focused on the purified protein, they are placed in a controlled environment in order to obtain the function information thereof; vivo research experiments focused on the activity of the protein in the cells or whole tissue, which can understand protein function places and the corresponding adjustment mechanism. Could help explore the biological function of the protein and the structure and function in vivo or in vitro protein engineering and protein modification. The mark of the protein variety of chemical methods and the use of trans-splicing intein tag. Former most of the relatively simple, but there are a lot of inadequacies, such specificity is not strong, the chemical label apt to interfere with the activity of the marker protein, higher protein concentration; latter protein labeling technology is an emerging, although it is overcomes the deficiencies of the chemical marker, but there are still a fusion protein instability, poor solubility, low activity and poor versatility urgent problem. Another way, on the basis of the protein trans-splicing introns looking for internal cysteine ??less activity was higher in the intron using site-directed mutagenesis to bypass the PCR methods to construct fracture intein obtained higher activity of the N-terminus (entire intein internal introns) cysteine, and by SDS-PAGE, Western Blot and other methods for detecting intein splicing activity, and then using the laboratory TM systems detect proteins within intron as the potential of the application of the marking means. Finally, choose the protein N-terminal-specific markers. The topics selected intein TerNdse-2, TX-S1, HaVol Pol, Msm DnaB-1, Arsp FB24 and PP-PhiEL ORF40 protein N-terminal tag. Msm DnaB Arsp FB24 3 type intein, the rest are a common type protein intron. TerNdse, TX-S1 (C1 / S), HaVol Po1 is Msm DnaB-1 and Arsp FB24 an amino serine, serine, alanine and glycine, is also very small of these intein internal cysteine ??conducive site-directed mutagenesis and subsequent mark. Original intron splicing activity first of these proteins to be detected, a weak connection active in vivo pMTerNdse-2, pMTX-S1 (C1 / S) has a high splicing activity (this laboratory has been studied in vivo and in vitro are high splicing activity), PMHP splicing activity of 100%, pMMD (C1 mutation into S) the weak splicing activity, pMAF no splicing activity. Then the right pMTerNdse-2 pMTX-S1 (C1 / S) and pMHP directed mutagenesis of amino acids, all of its internal cysteine ??through bypass PCR mutagenesis to serine, while the pMMD internal C1 recovery. Their splicing activity was detected by Western Blot. pMTerNdse-2, pMTX-S1 (C1 / S) and pMHP cysteine ??mutation no splicing activity, pMMD very high splicing activity. PMHP, its internal four cysteine ??were mutated to serine, we found that the second cysteine ??can determine its splicing activity, the use of site-directed mutagenesis method of its side-chain groups similar to the amino acid to be replaced First threonine and glycine replacement, got lucky the two internal splicing activity of cysteine-free HP mutant, pMHPG and pMHPT, laid the foundation for Di was a generic mark intein. Intein and laboratory peer determine S1, S0 and S11 three cleavage point to build pMTerNdse-2 pMMD, PMHP pMHPG and pMHPT corresponding S1, S0 and S11 vivo fracture intein . Its splicing activity was detected by Western Blot pMTerNdse-2, PMHP and pMHPG three fracture intein splicing activity, pMHPT-S1 has a high splicing activity; pMMD-SO splicing activity of 100%. Through the SWISS MODLE modeling, Protean peer from the new select other fracture sites, pMHP pMHPG and pMHPT selected 10 new cleavage sites for the F1-F10 5 pMMD re-select the cleavage site of the F1-F5. By Western Blot analysis in vivo splicing activity, the results show, PMHP-F2 and F8 high splicing activity; pMHPT only F2 splicing activity; pMMD only F4 have very high splicing activity. The to choose pMHP-F2 by the pMHP-F8, pMHPT-S1, pMHPT-F2 the pMMD-S0 pMMD-F4 and pMTX-S1 (C1 / S) several protein intron precursor protein construct containing the N-terminal or C-terminal the corresponding sequence of the expression plasmid of the precursor protein, maltose binding protein (maltose binding protein, MBP) and IN fusion protein consisting of a fusion protein composed of the precursor protein of the N-terminal: IC \u0026 thioredoxin (thioredoxin, T) for the C-terminal precursor protein. MBP-tag with the use of the precursor protein of the N-terminal can be used with the Amylose resin specifically binding to the C terminal of the precursor protein with the 6 × histidine tag can be combined with Ni-NTA resin, specificity and purified to be used for subsequent in vitro reaction precursor protein N-terminal and C-terminal precursor protein. The purified precursor protein of the N-terminal and C-terminal precursor protein at a molar ratio of 1:1 under the conditions of reaction, the activity of the splicing reaction by Western blot results showed that fracture protein the intron pMTX-S1 (C1 / S) and part pMMD-SO splicing the activity remaining intein splicing activity in vitro also require further experimental verification. Intron splicing activity in vitro fracture protein, we choose TXS1 TM system to detect the application of its protein N-terminal tag. The the TX-S1N built into the expression vector of the SUMO-containing PEDHC, the vitro purified the TX-S1N and TX-S1C protein, reacted for 4 hours the TX-S1N with the mercapto dye in the ratio of 1:5, was dialyzed through the dialysis bag off unreacted dye followed SUM0 enzyme and LX-S1N 1 hour of reaction, removal of the SUMO protein, L-TX-S1, and the TX-S1 C to react for 3 hours in vitro splicing buffer, SDS-PAGE detection, use of a wavelength of 346-442 nm UV observation photographed. This subject is not only on the previous studies, and also to overcome the inadequacies of the chemical synthesis and protein markers specific sites of the N-terminus of the protein marker. Controlled, specific target protein on the target protein markers laid the foundation and practical application. At the same time, we found a very interesting phenomenon: pMHP, and pMMD internal a non-conserved region of the cysteine ??intein activity can actually decide There is no literature reports, its chemical and molecular biological mechanism worth continue to study, I believe it is a good supplement of protein intron splicing theoretical knowledge. In addition, access to the two very high internal activity without cysteine ??intein mutants, although it did not get active in vitro fracture protein intron, but we will continue to study and want higher activity the multiple fracture intein, not only selected intein tag for the N-terminal, as well as the C-terminus of the protein markers or both ends of the mark are generic intein and a novel protein marked ways.

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CLC: > Industrial Technology > Chemical Industry > Pharmaceutical chemical industry > Production of organic compounds in drug > Elements of organic compounds in drug
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