Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.
Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.
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在乳腺癌细胞系中用仿射体标记突变雌激素受体。
DOI：
10.1016/j.bpj.2022.06.028
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发表时间：
2022
影响因子：
3.4
通讯作者：
Selvin,PaulR
中科院分区：
文献类型：
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作者：
Ren,Pin;Tiede,Christian;Fanning,SeanW;Adams,Thomas;Speirs,Valerie;Nelson,ErikR;Cheng,Changfeng;Moore,TerryW;Greene,GeoffreyL;Tomlinson,Darren;Selvin,PaulR
细胞内雌激素受体α（ERα）的突变与70%的乳腺癌有关。因此，对活癌细胞系中的各种突变体（L536 S、Y 537 S、D538 G）进行成像具有相当大的兴趣，特别是作为各种抗癌药物的作用。因此，我们开发了一种小的（13 kDa）Affimer，在荧光标记后，它能够通过毒素链球菌溶血素O在细胞膜上产生的临时孔来有效地标记ERα。通过噬菌体展示选择的Affimer主要标记Y 537 S突变体，并且可以区分L536 S和D538 G突变体之间的差异。绝大多数Affimer-ERα Y 537 S位于细胞核中，能够有效、不受限制地导航至其靶DNA序列，如单分子荧光所示。Affimer还可以区分选择性雌激素受体调节剂的作用。更一般地说，这是一个小的结合试剂的例子-Affimer蛋白质-可以插入到活细胞中，干扰最小，效率高，图像的内源性蛋白质。
Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent—an Affimer protein—that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.