荧光素酶互补

基本原理在验证植物蛋白互作时，荧光素酶互补实验(LuciferaseComplementationAssay,LCA)因其高灵敏度、可定量化、操作简单高效被广泛应用于植物学和动物学蛋白质互作研究目前,应用广泛的荧火素酶基因来源于北美萤火虫(fireflyluciferase),该基因编码550个氨基酸组成的荧火素酶蛋白(大小为62kDa)。实验中,荧光素酶蛋白被切成N端和C端2个功能片段,即NLuc(2–416AA)和CLuc(398–550AA)。在一个实验体系中,待检测的2个目标蛋白分别与NLuc和CLuc融合,如果2个目标蛋白相互作用,则荧火素酶的NLuc和CLuc在空间上会足够靠近并正确组装,从而发挥荧火素酶活性,即分解底物产生荧光。Abstract:Protein-Proteininteractionsplayimportantrolesinvariouseukaryoticbiologicalprocesses.Comparedtoothertechniquesmeasuringprotein-proteininteractionsinplants,theLuciferaseComplementationAssay(LCA),basedonAgrobacterium-mediatedtransientexpressioninNicotianabenthamiana,isasensitive,reliable,highlyquantitativeandlowbackgroundmethodthatcanbeeasilyscaledupforhigh-throughputinteractomestudies.Here,wedescribeaprotocolthatincludestwoalternativedatacollectionmethodstoqualitativeandquantitativeanalyseluminescenceorluminousintensitytodetectprotein-proteininteractionsinplantcells.

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