阿拉伯芥轉錄因子 ICE1 之重組蛋白表現及結構分析

Abstract

在阿拉伯芥中，Inducer of CBF Expression 1 (ICE1) 是一個 bHLH 家族轉錄因子。目前已經看到 ICE1 有十個交互作用的蛋白質，並且可被其他多個蛋白在不同位點進行轉譯後修飾，並參與許多不同生理功能。本研究從蛋白之結構的角度研究為何 ICE1 會在植物中扮演如此多重角色。二級結構預測軟體顯示 ICE1 有約 60% 的序列被預測為 '固有無序蛋白' 片段，這種蛋白常有許多不同交互作用蛋白，很有可能就是 ICE1 能夠在植物體中有多重角色及功能的原因。本研究首先以得到重組蛋白為目標，建立了 ICE1 重組蛋白的表現系統，成功在包涵體中表現出 ICE1 重組蛋白，將包涵體再溶解後，以多種不同的方式進行再摺疊及純化。樣品分別以圓二色光譜和動態光散射儀對其結構與化學性質作初步的分析及探討，發現其二級結構的組成會因溶液而產生變化，並且會形成不同大小的粒子，可能聚集形成聚合物或是蛋白凝集。當提高溶液鹽濃度時，我們透過圓二色光譜觀察到 ICE1 重組蛋白較易形成 α-螺旋的二級結構，且透過動態光散射儀觀察到粒子大小降低，顯示蛋白凝集程度減緩。這些結果暗示 ICE1有可能在環境改變時也產生結構上的改變。

Inducer of CBF Expression 1 (ICE1) is a MYC-like basic helix-loop-helix (bHLH) transcription factor in Arabidopsis thaliana. ICE1 interacts with or subjects to be post-translational modified by at least ten different proteins, and participates in different physiological functions. This study aims to understand why ICE1 can have so many functions from the structural point of view. Secondary structure analysis suggested that about 60% of the full length of ICE1 was likely to be intrinsic disordered protein region (IDPR). It is possible that the ability of ICE1 to interact with multiple proteins is due to its putative IDP nature. The production of recombinant ICE1 protein was established in this study. The recombinant protein was mainly produced as inclusion bodies. Isolated inclusion bodies were resolubilized and subjected to several refolding protocols in an attempt to obtain soluble and monomeric form for structural analysis. Soluble form of ICE1 recombinant protein was prepared using on-column refolding by affinity chromatography, and then analyzed by far-UV CD spectroscopy and dynamic light scattering. The secondary structure composition and particle size may vary in different conditions, which implied that it may form polymers or aggregation. High concentrations of NaCl might increase the α-helix forming ability as suggested by CD spectrum. Further, the smaller particle size revealed by DLS suggest a lower protein aggregation. These results implied that ICE1 has the ability to undergo structural changes in different environment.