双分子荧光互补技术BiFCBimolecular Fluorescence Complementation

 

双分子荧光互补技术是将荧光报告蛋白按照规则分成没有荧光的两段N-fragment及C-fragment,分别与诱饵蛋白和捕获蛋白融合,如果诱饵蛋白和捕获蛋白能发生相互作用,那么两段不完整的荧光报告蛋白片段就会形成完整的荧光报告蛋白,在激发光的激发下发出荧光。该技术可以用于检测蛋白-蛋白相互作用、药物筛选等,可用荧光显微镜、激光共聚焦显微镜、流式细胞仪等进行荧光细胞的实时观察、实时分选等。

 

 

1BiFC技术检测优势:
活细胞分析,直观、快速地判断目标蛋白在活细胞中的定位和相互作用;
可以用于研究2种或2种以上的蛋白质间相互作用;
具有很高的信噪比,弱蛋白相互作用也可以检测到(>7nm)。

2)可用于BiFC的荧光蛋白:
目前报道的用于BiFC检测的荧光蛋白有GFP、BFP、CFP、YFP、Venus、citrine、cerulean、mCherry等。Venus(EYFP的变体)是目前用于BiFC分析最多的荧光蛋白,因为其荧光强且背景敏感度降低,是BiFC分析理想的荧光蛋白。

 

常见的用于双分子荧光互补技术的荧光蛋白及拆分位点

 

3BiFC技术的应用:

ABiFC检测蛋白相互作用:

摘自Hox Proteins Display a Common and Ancestral Ability to Diversify Their Interaction Mode with the PBC Class Cofactors. Bruno Hudry, Sophie Remacle, Marie-Claire Delfini, René Rezsohazy, Yacine Graba, and Samir Merabet. PLoS Biol. 2012 Jun; 10(6): e1001351.

BBiFC检测细胞融合:

Validation of the BiFC approach to detect cell fusion.Two populations of COS-1 cells were transfected overnight with VN-Histone H3.1 and YC-Histone H3.1 respectively. The two populations of cells were then plated together one day prior to induction of fusion by PEG1500. (a–e) Typical BiFC fusion signals. Fusion signals (BiFC, green) were detected by fluorescence microscopy and images were obtained at high (a) and low magnification (d). Histone H3.1 was localized to the nucleus (b, Hoechst 33142 stain, blue; c, e, a merge with phase contrast). (f) Kinetic analysis of BiFC fusion signals. Two populations of COS-1 cells were transfected with plasmids encoding BiFC partners in the presence or absence of PEG1500. In addition, two populations of COS-1 cells were transfected with the same BiFC complex (i.e. VN-Histone H3.1+VN-Histone H3.1 or YC-Histone H3.1+YC-Histone H3.1). The number and location of cells per well with fusion signals were counted over a period of 24 h after fusion was induced. Three wells were counted for each condition. The mixture containing both BiFC partners induced to fuse via PEG showed the most dramatic increase in number of signals over time. In contrast, cell populations containing only one BiFC partner (i.e. VN- or YC-Histone H3.1) showed no formation of BiFC signals. (g) Time-lapse imaging of BiFC fusion signals. Cells were prepared as above and images were acquired after PEG-induced fusion at a frequency of 1/10 min over a period of 12 h. The upper panels show fusion signals and the lower panels show the same signals merged with a corresponding phase image. Scale bars in (a–g), 20 μm.

摘自Bimolecular fluorescence complementation analysis of eukaryotic fusion products. Ho-Pi Lin, Claudius Vincenz, Kevin W. Eliceiri, Tom K. Kerppola, and Brenda M. Ogle. Biol Cell. 2010 Aug 6; 102(Pt 9): 525–537.

CBiFC检测蛋白复合体在染色体上的定位:

Visualization of BiFC complexes on polytene chromosomes. (A) Diagram of BiFC complex formation on polytene chromosomes. The intensity of BiFC complex fluorescence at individual genomic loci is enhanced by the parallel alignment of hundreds of chromatids within the polytene chromosomes. (B) Examples of BiFC complex fluorescence (green) on polytene chromosomes stained using Hoechst (blue). The chromosomes were spread under acid-free conditions. The banding patterns of the polytene chromosomes stained using Hoechst are shown in the images to the right. Note the differences in chromosome spreading and extension that are necessary to map the localization of the BiFC complexes to individual genomic loci. Upper panel: an intact spread allowing mapping of a number of loci, including the two copies of the 55F locus, corresponding to the two homologues that were presumably separated during the squash procedure; Middle panel: a partial spread with broken arms, which are well extended and suitable for mapping a subset of the loci; Lower panel: a poorly extended spread on which the loci that are bound by BiFC complex cannot be easily identified. Scale bars: 10 µm. (C) Comparison of the localization of BiFC complexes and of the individual BiFC fusion proteins using acid-free (upper images) and conventional (middle and bottom images) squash protocols. Polytene chromosomes that prepared using the acid-free protocol have less sharp banding patters than those that are prepared using conventional squash protocols. Both dKeap1-CncC BiFC complexes and each of the fusion proteins bound the 55F locus. Both dKeap1 and CncC fusions bound many other loci, whereas the dKeap1-CncC BiFC complex bound the 55F locus with higher specificity. Scale bars: 5 µm.

摘自Visualization of the genomic loci that are bound by specific multiprotein complexes by bimolecular fluorescence complementation (BiFC) analysis on Drosophila polytene chromosomes. Huai Deng and Tom K Kerppola. Methods Enzymol. 2017; 589: 429–455.

DBiFC检测HIV病毒侵染宿主细胞:

Gag interactions with host proteins Staufen1 and IMP1 occur in the cytoplasm and at the plasma membrane of transfected HeLa and Jurkat T cells as determined by BiFC. (A) Top – schematic representation of BiFC method. Bottom – Rev-dependent Gag-VN and Gag-VC were co-transfected with pCMV-Rev in HeLa cells. At 24 hr post-transfection, cells were imaged by laser scanning confocal microscopy to detect BiFC. The white arrows indicate plasma membrane concentrated accumulations of Gag-Gag BiFC signals. (B) Gag-VN and Staufen1-VC (top panels) or Gag-VN and IMP1-VC (bottom panels) interactions identified by BiFC. BiFC signals for these interacting pairs were mainly detected in the cytoplasm (indicated by white arrows) and at or near the plasma membrane. (C) Interactions between Gag-VN with IMP1-KH(1-4)-VC (top) and with IMP1-RRM(1-2)-VC (bottom) as determined by BiFC analysis. Evidence for interaction is demonstrated by a green fluorescence signal. (D) The interaction between Gag-VN and Gag-VC (top) or Gag-VN and Staufen1-VC (bottom) was determined by BiFC in Jurkat T cells. Magnified sections demonstrate details on the shapes of BiFC signals/complexes. The size bars are equal to 10 μm.

摘自Live cell visualization of the interactions between HIV-1 Gag and the cellular RNA-binding protein Staufen1. Miroslav P Milev, Chris M Brown, and Andrew J Mouland.

Retrovirology. 2010; 7: 41.

BiFC技术检测蛋白-蛋白相互作用,广泛应用于植物、动物、微生物、病毒等的染色体、RNA、细胞融合等不同领域的研究。BiFC也可用于药物开发,首先选取疾病相关的重要蛋白,基于BiFC技术开发BiFC稳转细胞株,不同的药物处理后检测荧光信号就可筛选潜在的治疗药物,如DiscoverX公司开发了一系列GPCR相关的BiFC细胞株,用于筛选GPCR家族相关药物,具有良好的应用前景。

 

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