Construction,Expression and Purification of SUMO1-GST Fusion Protein-中国期刊网

摘要 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.

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Construction,Expression and Purification of SUMO1-GST Fusion Protein

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Construction,Expression and Purification of SUMO1-GST Fusion Protein

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