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| Type |
Poster Presentation |
| Area |
생명화학 |
| Room No. |
포스터발표장 |
| Time |
4월 21일 (금요일) 13:00~14:30 |
| Code |
BIO.P-291 |
| Subject |
The kinetics studies on protein trans-splicing reaction based on electrostatic potential of exteins |
| Authors |
이민형, 권영은*
동국대학교 의생명공학과, Korea
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| Abstract |
Protein trans-splicing (PTS) reaction is self-processing reaction mediated by a pair of split-inteins. PTS-based protein semi-synthesis is widely used for conjugation of various synthetic probes to target proteins in vivo and in vitro. As PTS reaction became a useful tool for various biological studies, it is important to understand the factors that affect the kinetics of PTS. Previously, there are a number of reports describe the roles of penultimate residues of inteins and the key amino acids of N- and C-exteins, usually the immediately flanking sequences, for ensuring the PTS activity. However, we also observed the PTS rates varied in large extend depending on the proteins that are used as exteins, even when the penultimate residues and immediate flanking sequences are well maintained. And there is no reasonable explanation offered for this variation yet. As there is an enormous complexity of all possible flanking sequences, we analyzed how the electrostatic potentials of flanking sequences affect PTS kinetics. For this study, we have chosen a fast reacting (t1/2 ~1 min) and naturally split-Npu DnaE intein as a model system. We prepared multiple pairs of split-inteins that carry exteins of various pI’s and analyzed the kinetics of in vitro PTS reactions. We observed that the higher pI value of N-intein flanking sequences (N-flanking) and lower pI value of C-intein flanking sequences (C-flanking) accelerated PTS reaction. We also confirmed this result using PTS reactions on live cells. A model cell surface protein was fused to N-intein and reacted with two fluorescent C-inteins. The C-intein carrying an extein with lower pI mediated faster labeling reaction. This study offers a good guideline in designing fast reacting PTS system. |
| E-mail |
sksdlalsgud@gmail.com |
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119th General Meeting of the KCS