Engineering and characterization of a superfolder green fluorescent protein
Engineering and characterization of a superfolder green fluorescent protein
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DOI：
10.1038/nbt1172
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发表时间：
2006-01-01
影响因子：
46.9
通讯作者：
Waldo, GS
中科院分区：
文献类型：
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作者：
Pédelacq, JD;Cabantous, S;Waldo, GS
绿色荧光蛋白（GFP）的现有变体在作为与其他蛋白质的融合蛋白表达时常常发生错误折叠。我们已经生成了一种折叠能力很强的GFP版本，称为“超级折叠”GFP，即使与折叠不良的多肽融合时也能很好地折叠。与“折叠报告”GFP（一种包含“第3轮”突变以及“增强型GFP”突变F64L和S65T的折叠增强型GFP）相比，超级折叠GFP对循环排列的耐受性提高，对化学变性剂的抵抗力更强，并且折叠动力学得到改善。将来自嗜热杆菌（Pyrobaculum aerophilum）的18种蛋白质分别与超级折叠GFP融合后在大肠杆菌细胞中表达，其荧光强度与总蛋白表达量成正比。相比之下，折叠报告GFP融合蛋白的荧光与被融合蛋白的有效折叠产量密切相关。X射线晶体结构分析有助于解释超级折叠GFP相对于折叠报告GFP折叠能力增强的原因。
Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.