A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms
A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms
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DOI：
10.1002/pro.18
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发表时间：
2009-01-01
期刊：
影响因子：
8
通讯作者：
Minor, Daniel L., Jr.
中科院分区：
文献类型：
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作者：
Hammon, Justus;Palanivelu, Dinesh V.;Minor, Daniel L., Jr.
绿色荧光蛋白 (GFP) 融合蛋白为鉴定表达良好、行为正常的膜蛋白提供了一种潜在的简便工具，可用于生化和结构研究。在这里，我们对来自 18 种细菌和古菌极端微生物的超过 300 种膜蛋白进行了 GFP 表达调查，这些生物体有望成为具有强大生物物理特性的膜蛋白的丰富来源。我们发现 GFP 融合荧光强度是过度表达潜力的极好指标。通过使用一套非 GFP 构建体和不同表达温度的后续优化方案，我们获得了 90% 通过 GFP 筛选的测试候选蛋白的 0.5-15 mg L-1 表达水平。对结果的评估表明，某些生物体可能是比其他生物体表达良好的膜蛋白的更好来源，密码子使用与表达宿主匹配的程度与成功率不相关，并且 GFP 筛选和表达优化的结合对于生产生化上易于处理的材料量至关重要。
Green fluorescent protein (GFP) fusion proteins provide a potentially facile tool for identification of well expressed, properly behaved membrane proteins for biochemical and structural study. Here, we present a GFP-expression survey of >300 membrane proteins from 18 bacterial and archaeal extremophiles, organisms expected to be rich sources of membrane proteins having robust biophysical properties. We find that GFP-fusion fluorescence intensity is an excellent indicator of over-expression potential. By employing a follow-up optimization protocol using a suite of non-GFP constructs and different expression temperatures, we obtain 0.5-15 mg L-1 expression levels for 90% of the tested candidate proteins that pass the GFP screen. Evaluation of the results suggests that certain organisms may serve as better sources of well-expressed membrane proteins than others, that the degree to which codon usage matches the expression host is uncorrelated with success rate, and that the combination of GFP screening and expression optimization is essential for producing biochemically tractable quantities of material.