简介:Sumoylationisanimportantproteinmodificationdiscoveredrecently.SUMO(smallubiquitin-relatedmodifier)pathwayregulatestheproteinstabilityandtranscriptionalactivitywitha12-kDasmallmolecularprotein,SUMO,ligatedtothetargetprotein.ThepurificationofSUMOproteinsisakeysteptorevealtheirfunction.ThepurposeofthisstudywastoconstructtherecombinantSUMO1geneclonedtoapGEX-4T-1vectortoexpressandpurifytheSUMO1-GSTfusionproteininEscherichiacoli.First,thefulllengthDNAsequenceofSUMO1genewasamplifiedbyPCRandwasligatedtopMD18-Tvector.ThentheSUMO1genewassubclonedtopGEX-4T-1prokaryoticexpressionvectorbetweenBamHIandXhoIsites,andtransformedinEscherichiacoliDH5αcells.Therightcolonieswereidentifiedbyrestrictiveenzymedigestionandsequencing.ThecorrectrebombinantplasmidofpGEX-4T-1-SUMO1wastransformedinEscherichiacoliBL21cellsandtheninducedbyIPTG(isopropyl-β-D-1-thiogalacto-pyranoside)toexpresstheSUMO1-GSTfusionprotein.ThehighlypurifiedSUMO1-GST(glutathioneS-transferase)fusionproteinwasobtainedbyaffinitychromatography.Finally,thepropertiesofSUMO1-GSTfusionproteinwereconfirmedbyCoomassiebrilliantbluestrainandWesternblotanalysis.TherecombinantplasmidofpGEX-4T-1-SUMO1wassuccessfullyconstructed,andSUMO1-GSTfusionproteinsweresuccessfullyexpressed.
简介:Thelackofefficientandnon-toxicgenedelivery,preferablywithnon-viralDNAvectors,isgenerallyregardedasamajorlimitationforgenetherapy.Inthisstudy,awheathistoneH4genewasclonedfromTriticumaestivum,sequenced,modifiedandexpressedinE.coli.ThewheathistoneH4geneandreconstructedH4TLgeneencodedwheathistoneH4andarecombinantproteinof141aminoacidswithanapproximatemolecularweightof15500.GelelectrophoresismobilityshiftassaysdemonstratedthatthepurifiedproteinhadhighaffinityforDNA.Mostsignificantly,thecomplexofplasmidpEGFP/C1withH4TLwastransfectedwithincreasedefficiencyintoMCF-7,HO8910,LNCap,A549andHeLacellsinvitro.Theseresultsdemonstratethatthetargetingofnon-viralvectorstotumor-specificreceptorsprovidesacheap,simpleandhighlyefficienttoolforgenedelivery.