PERSONAL DE APOYO
BAYARDO Mariela Paula
artículos
Título:
ProductionoftheMainCeliacDiseaseAutoantigenbyTransientExpressioninNicotianabenthamiana
Autor/es:
VANESA MARIN VIEGAS; GONZALO ACEVEDO; MARIELA BAYARDO; FERNANDO CHIRDO
Revista:
Frontiers in Plant Science
Editorial:
ItalianNationalAgencyforNew Technologies,EnergyandSustainable EconomicDevelopment,Italy
Referencias:
Año: 2015 vol. 6 p. 1 - 11
Resumen:
CeliacDisease(CD)isaglutensensitiveenteropathythatremainswidelyundiagnosedandimplementationofmassivescreeningtestsisneededtoreducethelongtermcomplicationsassociatedtountreatedCD.ThemainCDautoantigen,humantissuetransglutaminase(TG2),isachallengeforthedifferentexpressionsystemsavailablesinceitscross-linkingactivityaffectscellularprocesses.Plant-basedtransientexpressionsystemscanbeanalternativefortheproductionofthisprotein.Inthiswork,atransientexpressionsystemfortheproductionofhumanTG2inNicotianabenthamianaleaveswasoptimizedandreactivityofplant-producedTG2inCDscreeningtestwasevaluated.First,asubcellulartargetingstrategywastested.Cytosolic,secretory,endoplasmicreticulum(C-terminalSEKDELfusion)andvacuolar(C-terminalKISIAfusion)TG2versionsweretransientlyexpressedinleavesandrecombinantproteinyieldsweremeasured.ER-TG2andvac-TG2levelswere9-to16-foldhigherthantheircytosolicandsecretorycounterparts.Assecondstrategy,TG2variantswereco-expressedwithahydrophobicelastin-likepolymer(ELP)constructencodingfor36repeatsofthepentapeptideVPGXGinwhichtheguestresidueXwereVandFinratio8:1.Proteinbodies(PB)wereinducedbytheELP,withaconsequenttwo-fold-increaseinaccumulationofbothER-TG2andvac-TG2.Subsequently,ER-TG2andvac-TG2wereproducedandpurifiedusingimmobilizedmetalionaffinitychromatography.PlantpurifiedER-TG2andvac-TG2wererecognizedbythreeanti-TG2monoclonalantibodiesthatbinddifferentepitopesprovingthatplant-producedantigenhasimmunochemicalcharacteristicssimilartothoseofhumanTG2.Lastly,anELISAwasperformedwithseraofCDpatientsandhealthycontrols.Bothvac-TG2andER-TG2werepositivelyrecognizedbyIgAofCDpatientswhiletheywerenotrecognizedbyserumfromnon-celiaccontrols.Theseresultsconfirmedtheusefulnessofplant-producedTG2todevelopscreeningassays.Inconclusion,thecombinationofsubcellularsortingstrategywithco-expressionwithaPBinducingconstructwassufficienttoincreaseTG2proteinyields.Thistypeofapproachcouldbeextendedtootherproblematicproteins,highlightingtheadvantagesofplantbasedproductionplatforms.
