酵母
蛋白质工程
定向进化
变性(裂变材料)
突变体
蛋白质稳定性
热稳定性
热休克蛋白
流式细胞术
合成生物学
酿酒酵母
表面蛋白
表面工程
生物
化学
细胞生物学
生物化学
基因
材料科学
计算生物学
分子生物学
纳米技术
酶
有机化学
病毒学
核化学
作者
Michael W. Traxlmayr,Eric V. Shusta
出处
期刊:Methods in molecular biology
日期:2017-01-01
卷期号:: 45-65
被引量:24
标识
DOI:10.1007/978-1-4939-6857-2_4
摘要
Yeast surface display is a powerful protein engineering technology that has been used for many applications including engineering protein stability. Direct screening for improved thermal stability can be accomplished by heat shock of yeast displayed protein libraries. Thermally stable protein variants retain binding to conformationally specific ligands, and this binding event can be detected by flow cytometry, facilitating recovery of yeast clones displaying stabilized protein variants. In early efforts, the major limitation of this approach was the viability threshold of the yeast cells, precluding the application of significantly elevated heat shock temperatures (>50 °C) and therefore limited to the engineering of intrinsically unstable proteins. More recently, however, techniques for stability mutant gene recovery between sorting rounds have obviated the need for yeast growth amplification of improved mutant pools. The resultant methods allow significantly higher denaturation temperatures (up to 85 °C), thereby enabling the engineering of a broader range of protein substrates. In this chapter, a detailed protocol for this stability engineering approach is presented.
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