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与弓形虫微线体蛋白互作的宿主蛋白的筛选和鉴定.docx

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与弓形虫微线体蛋白互作的宿主蛋白的筛选和鉴定 Bowmaniamicrotiisaparasiticprotozoanthatinfectsawiderangeofmammals,includinghumans.Thisorganismisresponsibleforcausingasevereandoftenfataldiseaseknownastoxoplasmosis.Thekeytounderstandingthepathogenesisofthisdiseaseliesintheinteractionsbetweentheparasiteandthehost.Inparticular,theidentificationofhostproteinsthatinteractwiththeBowmaniamicrotimicrotubuleprotein,acrucialcomponentoftheparasitecelldivisionmachinery,isofcriticalimportance.Inthisreview,wewilldiscussthecurrentapproachesforscreeningandidentifyinghostproteinsthatinteractwiththeBowmaniamicrotimicrotubuleprotein. OnepromisingapproachforidentifyinghostproteinsthatinteractwiththeBowmaniamicrotimicrotubuleproteinistheyeasttwo-hybridsystem.Thissystemallowsfortheidentificationofprotein-proteininteractionsinvivo,andhasbeenusedsuccessfullytoidentifyhostproteinsthatinteractwithotherpathogenicmicroorganisms.InthecontextofBowmaniamicroti,thissysteminvolvesexpressingthemicrotubuleproteinasthebaitinayeaststrainthatcarriesalibraryofhostproteinsfusedtotheGal4activationdomain.Interactionsbetweenthebaitandpreyproteinsactivatetheexpressionofreportergenes,enablingtheidentificationofinteractinghostproteins.ThisapproachhasalreadybeenusedtoidentifyseveralhostproteinsthatinteractwiththeBowmaniamicrotimicrotubuleprotein,includingalpha-tubulin,beta-tubulin,andgamma-tubulin. AnotherapproachforidentifyinghostproteinsthatinteractwiththeBowmaniamicrotimicrotubuleproteinisaffinitypurificationcombinedwithmassspectrometry.Inthisapproach,themicrotubuleproteinisexpressedinahostcellline,andtaggedwithaspecificproteinpurificationtag,suchastheaffinitytagGFP.Thetaggedproteinisthenpurifiedfromthehostcelllysateusingaspecificantibodyorresin,andthepurifiedproteinisanalyzedbymassspectrometrytoidentifyinteractinghostproteins.Thisapproachhastheadvantageofidentifyinginteractingproteinsunderphysiologicalconditions,butitrequiresahighdegreeofspecificityoftheaffinitytag,aswellastheavailabilityofasuitableantibodyorresinforpurification.Are