双分子荧光互补技术(BiFC)资料下载.pdf

1、（765）496-1971Fax:（765）494-1414E-mail:hu1purdue.eduPresentation OutlineProtein-Protein InteractionsThe principle of BiFCThe Applications of BiFCPart 1 Protein-Protein Interactions 蛋白质相互作用（蛋白质相互作用（Protein-Protein Interactions）Proteinprotein interactions occur when two or more proteins bind together,of

2、ten to carry out their biological function.DNA replicationSignal transduction研究蛋白质相互作用的方法1.酵母双杂交技术（Yeast tow-hybrid ）2.噬茵体展示技术（Phage display technology）3.表面等离子共振技术（Surface Plasma resonance technique）4.荧光能量转移技术（Fluorescence energy transfer,FRET）5.抗体与蛋白质阵列技术（Antibody and protein array technology）6.免疫共

3、沉淀技术（Immunoprecipitation）7.pull-down技术（pull-down technology）8.双分子荧光互补（Bimolecular fluorescence complementation,BiFC）Part 2 The principle of BiFCGFP的发光机制GFP由238个氨基酸分子组成，分子量为26.9 kDa。来源于水母的野生型GFP在395 nm和475 nm分别有主要和次要的激发峰，它的发射峰在509 nm，处于可见光谱的绿色区域；来源于海肾的GFP只在498 nm有单个激发峰。GFP是典型的桶形结构，包含折叠和螺旋，将荧光基团包含在其中。

4、严密的桶形结构保护着荧光基团，防止它被周围环境淬灭，内部面向桶形的侧链诱导Ser65Tyr66Gly67三肽环化，导致荧光基团形成。双分子荧光互补（BiFC）荧光！一种直观、快速地判断目标蛋白在活细胞中的定位和相互作用的新技术。在GFP 的两个片层之间的环结构（loop）上有许多特异位点可以插入外源蛋白而不影响GFP的荧光活性，利用该荧光蛋白家族的这一特性，将荧光蛋白分割成两个不具有荧光活性的分子片段，再分别与目标蛋白连接。Bimolecular Fluorescence Complementation（BiFC）Twofragments（YNandYC）of theyellowfluores

5、centprotein（YFP）arefusedto twoputativeinteractionpartners（Aand B）Aninteractionbetweentheproteinsfacilitatesassociationbetweenthefragmentstoproducea bimolecularfluorescentcomplexYNand YCdo notinteracton theirownBiFC的优点（Pros）1.Relevant biological context2.Direct visualization3.Sensitivity4.Spatial res

6、olution5.No specialized equipment6.No structural information needed7.Multiple applicationsBiFC的缺点（Cons）1.Real-time detection2.Irreversible BiFC formation3.Independent fluorescent protein fragment associations4.Altering protein structure and steric hindrance5.Obligate anaerobes6.Use of fusion protein

7、s7.Temperature dependence8.Exact interaction relationship unknownBiFC系统的扩展1、Multicolor BiFC（多色双分子荧光互补）2、TriFC（三分子荧光互补）3、BiFC-FRETMulticolor BiFC（1）Many proteins have alternative interactionpartners ineachcell,manyaremutuallyexclusive and result in competition for sharedinteractionpartnersinthecell.（

8、2）Based oncomplementation betweenfragments of fluorescent proteins with differentspectralcharacteristics（3）Allows simultaneous visualization ofmultipleproteincomplexesinthesamecell（4）Enables analysis of the competitionbetweenalternativeinteractionpartnersforcomplexformationTriFCBiFC-FRETDevelopment

9、of a BiFC-based FRET assay.ShyuY J et al.PNAS 2008;105:151-1562008 by National Academy of SciencesThe dos and donts（1）荧光片段和目标蛋白质之间最好加1个连接肽，以避免蛋白质空间位阻所导致的片段间不能相互靠近。常用的连接肽氨基酸序列有 RSIAT,RPACKIPNDLKQKVMNH和GGGGS等；（2）温度对片段间互补影响很大，可以有两种解决方案。一是在室温或低于室温（25）下培养细菌或细胞，二是在生理条件下培养细菌或细胞，使融合蛋白正常表达，然后将培养物低温处理 1 到 2 h

10、 或接着于室温培养 1d；（3）建立阴性对照，以便更加确信BiFC信号反映的是蛋白质之间的相互作用。阴性对照通常是将相互作用的蛋白进行突变，降低或缺失其相互作用能力，再采用相同策略的BiFC系统检测。Workflow For BiFCSelection of fusion protein expression systemDetermination of fusion sitesDesigning linkersCreating proper plasmid expression vectorsSelection of appropriate cell culture systemSelect

11、ion of appropriate controlsCell transfectionVisualization and analysisCareful selection of the fluorescent protein is important,as different fluorescent proteins require different cellular environments.For example,GFP can be used in E.coli cells,while YFP is used in mammalian cells.A linker is a sho

12、rt amino acid sequence that tethers the fluorescent reporterproteinfragmenttotheproteinofinterest,formingthefusionprotein.When designing a linker sequence,one must ensure that the linker is sufficientlysoluble and long to provide the fluorescent protein fragments with flexibility andfreedom of movem

13、ent so that the fragment and its partner fragment will collidefrequently enough to reconstitute during the interaction of their respective fusedproteins.Although it is not documented,it is possible that the length or the sequence ofthelinkermay influencecomplementationofsomeproteins.N-terminal fragm

14、ent fused at the N-terminal protein 1+C-terminal fragment fused at the N-terminal protein 2N-terminal fragment fused at the N-terminal protein 1+C-terminal fragment fused at the C-terminal protein 2N-terminal fragment fused at the C-terminal protein 1+C-terminal fragment fused at the N-terminal prot

15、ein 2N-terminal fragment fused at the C-terminal protein 1+C-terminal fragment fused at the C-terminal protein 2C-terminal fragment fused at the N-terminal protein 1+N-terminal fragment fused at the N-terminal protein 2C-terminal fragment fused at the N-terminal protein 1+N-terminal fragment fused a

16、t the C-terminal protein 2C-terminal fragment fused at the C-terminal protein 1+N-terminal fragment fused at the N-terminal protein 2C-terminal fragment fused at the C-terminal protein 1+N-terminal fragment fused at the C-terminal protein 2When designing plasmid vectors to express the proteins of interest,the construct must be able to express proteins that are able to form fusion proteins with fluorescent protein fragments withou