蛋白質(zhì)作為藥物,、生物制劑和催化劑被廣泛應(yīng)用，其穩(wěn)定性對(duì)于生物技術(shù)應(yīng)用具有重要影響,。通常,，蛋白質(zhì)的穩(wěn)定性可以通過定向進(jìn)化、序列同源比對(duì)和理性設(shè)計(jì)來完成,。上述方法都有各自優(yōu)勢(shì)和缺點(diǎn),，在實(shí)際應(yīng)用中方法的適用性和成功率通常依賴于蛋白質(zhì)體系本身。
近日,，中國科學(xué)院青島生物能源與過程研究所仿真模擬團(tuán)隊(duì)設(shè)計(jì)出一套計(jì)算機(jī)虛擬篩選結(jié)合分子生物學(xué)實(shí)驗(yàn)方法,，成功提高了枯草芽孢桿菌藍(lán)色熒光蛋白質(zhì)YtVALOV的穩(wěn)定性。該方法主要包括如下步驟：1.采用計(jì)算機(jī)模擬對(duì)可能的熱穩(wěn)定突變進(jìn)行篩選,；2.對(duì)篩選出較重要突變體進(jìn)行實(shí)驗(yàn)驗(yàn)證,；3.用計(jì)算模擬對(duì)實(shí)驗(yàn)確認(rèn)的位點(diǎn)及其周圍進(jìn)行氨基酸再優(yōu)化；4.對(duì)優(yōu)化的突變位點(diǎn)進(jìn)行實(shí)驗(yàn)表征,；5.對(duì)獲取的穩(wěn)定突變進(jìn)行組合,。實(shí)驗(yàn)中，采用該方法成功將YtVALOV的Tm值提高了31°C（圖1）,。
該方法的優(yōu)勢(shì)在于不受蛋白質(zhì)體系本身限制,，理論上可以用來提高任何具有三維結(jié)構(gòu)的蛋白質(zhì)的穩(wěn)定性，對(duì)穩(wěn)定性差的蛋白質(zhì)制劑改性具有重要意義,。該研究成果已經(jīng)在線發(fā)表于Plos Computational Biology,。（生物谷 Bioon.com）
圖1.YtVA LOV熒光強(qiáng)度隨溫度的變化曲線
生物谷推薦的英文摘要
PLoS Comput Biol doi:10.1371/journal.pcbi.1003129
Engineering a More Thermostable Blue Light Photo Receptor Bacillus subtilis YtvA LOV Domain by a Computer Aided Rational Design Method Xiangfei Song equal contributor.
Yefei Wang equal contributor, Zhiyu Shu, Jingbo Hong, Tong Li, Lishan Yao
The ability to design thermostable proteins offers enormous potential for the development of novel protein bioreagents. In this work, a combined computational and experimental method was developed to increase the Tm of the flavin mononucleotide based fluorescent protein Bacillus Subtilis YtvA LOV domain by 31 Celsius, thus extending its applicability in thermophilic systems. Briefly, the method includes five steps, the single mutant computer screening to identify thermostable mutant candidates, the experimental evaluation to confirm the positive selections, the computational redesign around the thermostable mutation regions, the experimental reevaluation and finally the multiple mutations combination. The adopted method is simple and effective, can be applied to other important proteins where other methods have difficulties, and therefore provides a new tool to improve protein thermostability.
