Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide 英文参考文献.docVIP

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Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide 英文参考文献.doc

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Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide 英文参考文献

RobustExpressionandSecretionofXylanase1in ChlamydomonasreinhardtiibyFusiontoaSelection GeneandProcessingwiththeFMDV2APeptide BethA.Rasala1,PhilipA.Lee1,2,ZhouxinShen1,StevenP.Briggs1,MichaelMendez2., StephenP.Mayfield1*. 1TheSanDiegoCenterforAlgaeBiotechnologyandDivisionofMolecularBiology,UniversityofCaliforniaSanDiego,SanDiego,California,UnitedStatesofAmerica, 2SapphireEnergy,SanDiego,California,UnitedStatesofAmerica Abstract Microalgaehaverecentlyreceivedattentionasapotentiallow-costhostfortheproductionofrecombinantproteinsand novel metabolites. However, a major obstacle to the development of algae as an industrial platform has been the poor expressionofheterologousgenesfromthenucleargenome.Herewedescribeanuclearexpressionstrategyusingthefoot- and-mouth-disease-virus 2A self-cleavage peptide to transcriptionally fuse heterologous gene expression to antibiotic resistanceinChlamydomonasreinhardtii.Wedemonstratethatstrainstransformedwithble-2A-GFParezeocin-resistantand accumulatehighlevelsofGFPthatisproperly‘cleaved’attheFMDV2Apeptideresultinginmonomeric,cytosolicGFPthat is easily detectable by in-gel fluorescence analysis or fluorescent microscopy. Furthermore, we used our ble2A nuclear expressionvectortoengineertheheterologousexpressionoftheindustrialenzyme,xylanase.Wedemonstratethatlinking xyn1expressiontoble2Aexpressiononthesameopenreadingframeledtoadramatic(,100-fold)increaseinxylanase activityincellslysatescomparedtotheunlinkedconstruct.Finally,byinsertinganendogenoussecretionsignalbetween theble2Aandxyn1codingregions,wewereabletotargetmonomericxylanaseforsecretion.Thenovelmicroalgaenuclear expressionstrategydescribedhereenablestheselectionoftransgeniclinesthatareefficientlyexpressingtheheterologous gene-of-interestandshouldprovevaluableforbasicresearchaswellasalgalbiotechnology. Citation:RasalaBA,LeePA,ShenZ,BriggsSP,MendezM,etal.(2012)RobustExpressionandSecretionofXylanase1inChlamydomonasreinhardtiibyFusiontoa SelectionGeneandProcessingwiththeFMDV2APeptide.