Dissertation > Excellent graduate degree dissertation topics show

Kinetic Analysis of Trans-Splicing of Novel Split-Inteins in Vitro

Author: LiMengMeng
Tutor: MengQing；LiuXiangQin
School: Donghua University
Course: Biochemistry and Molecular Biology
Keywords: Fracture intein Trans-splicing Dynamics In vitro reaction
CLC: Q51
Type: Master's thesis
Year: 2010
Downloads: 23
Quote: 0
Read: Download Dissertation

Abstract

Intein (intein) is embedded in a host protein in the inserted sequence, able to self cut down in the process of maturation of host proteins, while the both sides of the sequence - protein exon (exteins) connected by a peptide bond is formed Mature proteins. This maturation process of the protein known as cis-splicing proteins (protein cis-splicing). On fracture intein (split-intein) breaks at specific sites in the protein interior region of intron, and the formation of the N-terminal protein (IN) and C-terminal protein (Ic), respectively, by the genome distant two open reading frame (open reading frame, ORF) encoding. In the post-translational maturation process, IN, and Ic recognize each other, the reconstruction of the catalytic active center-mediated trans-splicing protein (protein trans-splicing). Through the the artificial modification fracture intein cleavage sites, so close to the intein N-terminus or C-terminus, i.e. the formation of a new fracture intein. Breaking intein-mediated protein trans-splicing technology in protein purification, gene therapy, a cyclic peptide production and macromolecular complex protein production has yielded some results. On this basis, the new fracture intein expanded fracture intein application, the marker in the target protein's N-terminus or C-terminus of the potential of the first chemical group. Before the application of a comprehensive understanding of enzyme kinetics of this reaction, and thus, the the new fracture protein intron splicing dynamics research is very important. The subject to select a new fracture intein SX-S1, TX-S1, SX-S11 and TE3-S11 for splicing kinetics of. S1 fracture intein N terminal in the present study contains only 11 amino acids, the S11 type fracture intein containing only C terminal 6 amino acids, easy chemical synthesis, may be applied to the N-terminus of the recombinant protein or drug or C-terminal fluorescein, biotin tags. Construct containing only the expression plasmid of the precursor protein or the C terminal of the N end of the corresponding sequence of the precursor protein of maltose binding protein (maltose binding protein, MBP) fusion protein consisting of IN N end of the precursor protein; IC with thioredoxin ( thioredoxin, T) consisting of C terminal fusion protein precursor protein. N end of the precursor protein with MBP pins with Amylose Resin-specific binding, specific binding of the C end of the precursor protein with the 6 × histidine tag with a Ni-NTA resin, and purified to be used in the subsequent reaction in vitro the the precursor protein N-terminal and C-terminal precursor protein. Purified precursor protein and the C terminal of the N terminal of the precursor protein at a molar ratio of 1:1 carried out under the conditions of the reaction, the splicing reaction activity is detected by Western blot results showed that the fracture intein sub-SX-S1, TX- S1, SX-S11 and TE3-S11, both in vitro splicing activity. Combination of two of the the precursor protein N-terminal and C-terminal precursor protein in vitro cross-reactivity, addition to the TX-S1N/SX-S1C combinations, other combinations there was no cross-reactivity to multiple fracture protein intron a target protein multi-marker or mark the foundation of multiple target proteins. Type of DTT concentration, temperature, concentration of sodium chloride and the pH value of the condition, the explore breaking intein splicing optimum conditions for the results show that the addition to the reaction system in the presence of DTT, the splicing efficiency is decreased or even no splicing reaction, the remaining conditions of the splicing reaction was less affected. When the precursor protein and the C terminal of the N terminal of the precursor protein molar ratio of 10:1, the kinetic analysis of experimental results showed that the fracture intein sub-SX-S1 the TX-S1, SX-S11 and TE3-S11 in vitro splicing efficiency reached to 96% ~ 50% ~ 93% and ~ 87%, splicing rate constant (2.68 ± 0.39) x 10-4 s-1 (2.19 ± 0.33) × 10-3 s-1, (3.94 ± 0.91) × 104 s-1 and (3.43 ± 0.59) × 10-4 s-1. The new fracture protein intron position-specific protein markers of potential applications, the protein marker is no longer confined to traditional amino acid-specific marker. A new fracture protein introns in protein engineering, protein drug development and protein structure and function studies have a very wide range of applications.

Kinetic Analysis of Trans-Splicing of Novel Split-Inteins in Vitro

Abstract

Related Dissertations