The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins
The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins
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DOI：
10.1007/s004240100619
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发表时间：
2001-09-01
影响因子：
4.5
通讯作者：
Beam, KG
中科院分区：
文献类型：
--
作者：
Stroffekova, K;Proenza, C;Beam, KG
据报道，FLASH-EDT2 - 4 ',5 ' -bis（1,3,2-二硫胂-2-基）荧光素-（1,2-乙二硫醇）(2)-仅在与特定的四胱氨酸Motif （CCXXCC，“Cys(4)”）高亲和力结合后才会发出荧光，从而为体内标记重组蛋白提供了一种技术（Griffin等）。科学281:269 - 272)。我们尝试使用FLASH-EDT2作为骨骼肌中二氢吡啶受体（DHPR） II-III环的位点特异性标记。在异常肌管（缺乏内源性α (1S)）中表达后，α (1S)突变为II-III环中含有CCRECC，能够产生l型钙电流并介导骨型兴奋-收缩（EC）偶联，但FLASH-EDT2标记显示与未转染的异常肌管没有差异。转染含Cys(4)钙调素的HeLa-S3细胞比未转染的细胞具有更强的荧光性，而CHO-K和HEK 293细胞在转染和未转染的细胞之间的差异不太明显。由于FLASH- edt2处理后未转染细胞的荧光显著增强，提示FLASH可能与内源性含半胱氨酸蛋白结合。在试管实验中，将FLASH-EDT2加入到肌管或细胞系的蛋白匀浆中，观察到FLASH-EDT2荧光，证实了这一发现。用共价修饰半胱氨酸残基的coumarine马来酰亚胺（CPM）预处理细胞或细胞匀浆可消除增强的荧光。因此，在与引入的Cys(4)基序和内源性含半胱氨酸蛋白结合后，FLASH荧光似乎都会增强。因此，FLASH-EDT2可能仅用于标记那些表达水平非常高的重组蛋白。
FLASH-EDT2 - 4 ' ,5 ' -bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)(2) - has been reported to fluoresce only after binding with high affinity to a specific tetracysteine Motif (CCXXCC, "Cys(4)") and thus to provide a technique for labeling recombinant proteins in vivo (Griffin et al. Science 281:269-272). We have attempted to use FLASH-EDT2 as a site-specific label of the II-III loop of the dihydropyndine receptor (DHPR) in skeletal muscle. Upon expression in dysgenic myotubes (which lack endogenous alpha (1S)), an alpha (1S) mutated to contain CCRECC in the II-III loop was able to produce L-type calcium currents and to mediate skeletal-type excitation-contraction (EC) coupling, but FLASH-EDT2 labeling revealed no difference from non-transfected dysgenic myotubes. HeLa-S3 cells transfected with Cys(4)-containing calmodulin were significantly more fluorescent than non-transfected cells, whereas the difference between transfected and non-transfected cells was less apparent for CHO-K and HEK 293 cells. Because the fluorescence of non-transfected cells increased substantially after treatment with FLASH-EDT2, it suggested the possibility that FLASH binds to endogenous cysteine-containing proteins. This finding was confirmed in cuvette experiments in which FLASH-EDT2 fluorescence was observed after FLASH-EDT2 was added to protein homogenates from myotubes or cell lines. The enhanced fluorescence was abolished by pretreatment of cells or cell homogenates with coumarine maleimide (CPM), which modifies cysteine residues covalently. Thus, enhanced FLASH fluorescence appears to occur both after binding to an introduced Cys(4) motif and to endogenous, cysteine-containing proteins. Therefore, FLASH-EDT2 may be useful only for labeling those recombinant proteins that express at a very high level.