FACS-optimized mutants of the green fluorescent protein (GFP)
FACS-optimized mutants of the green fluorescent protein (GFP)
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DOI：
10.1016/0378-1119(95)00685-0
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发表时间：
1996-07-01
期刊：
影响因子：
3.5
通讯作者：
Falkow, S
中科院分区：
文献类型：
--
作者：
Cormack, BP;Valdivia, RH;Falkow, S
我们在大肠杆菌中构建了一个由严格调控的诱导型启动子表达的突变gfp基因（编码绿色荧光蛋白，GFP）文库。我们在发色团Ser-Tyr-Gly序列侧翼的20个氨基酸中在氨基酸65-67处引入随机氨基酸（aa）取代。然后，我们使用荧光激活细胞分选（FAGS）来选择GFP的变体，当在488 nm处激发时，这些变体的荧光强度比野生型（wt）高20至35倍。序列分析揭示了GFP中的三种类型的氨基酸取代。这三类突变蛋白的最大激发峰都发生了很大的偏移。在大肠杆菌中，突变体蛋白的折叠比野生型GFP的折叠更有效。这两个特性有助于大大增加（100倍）的荧光强度，使突变体可用于许多应用。
We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FAGS) to select variants of GFP that fluoresce between 20- and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima, In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.