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HUBEINORMALUNIVERSITYREPORTERXIANGRUCHENKEYLABORATORYOFSTRUCTUREANDFUNCTIONOFPROTEINCOLLEGEOFLIFESCIENCELITERATUREREPORTCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCECONTENT1HIGHLEVELEXPRESSIONOFTHENATIVEBARLEYAMYLASE/SUBTILISININHIBITORINPICHIAPASTORIS2AMANUALFORTHEPREPARATIONANDTRANSFORMATIONOFPICHIAPASTORISSPHEROPLASTSCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCEHIGHLEVELEXPRESSIONOFTHENATIVEBARLEYAMYLASE/SUBTILISININHIBITORINPICHIAPASTORISINTRODUCTION/PROGRESS1MATERIALSANDMETHODS2RESULTS3DISCUSSION4CONTENTBACKCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE1INTRODUCTION/PROGRESS1PREVIOUSLYMOSTPLANTPROTEINSEXPRESSEDINPPASTORISRESULTEDINLOWYIELDSCEREGHINOANDCREGG,20002BASITHEHORDEUMVULGAREAMYLASE/SUBTILISININHIBITORHASPREVIOUSLYBEENEXPRESSEDINPPASTORIS,BUTYIELDSWEREVERYLOWBNSAGERETAL,20033PICHIAPASTORISISONEOFTHEEUKARYOTICEXPRESSIONHOSTSTHATHAVEBEENDEVELOPEDINTOAHIGHLYSUCCESSFULEXPRESSIONSYSTEMMACAULEYPATRICKETAL,2005COPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE1INTRODUCTION/PROGRESS4HOWEVER,WITHINTHELASTCOUPLEOFYEARS,PLANTPROTEINSHAVESUCCESSFULLYBEENEXPRESSEDINPPASTORISATHIGHLEVELSFIERENSETAL,2004YONEYAMAETAL,2007PANETAL,20075INTHISSTUDYWEPRESENTSUCCESSFULHIGHLEVELEXPRESSIONOFTHEHORDEUMVULGAREAMYLASE/SUBTILISININHIBITORBASIINPPASTORISFROMJOURNALOFBIOTECHNOLOGY,20086INTHISPAPERAHIGHLEVELEXPRESSIONSYSTEMFORTHEBIFUNCTIONALAMYLASE/SUBTILISININHIBITORFROMBARLEYISPRESENTEDCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE1INTRODUCTION/PROGRESS7COMPAREDTOPREVIOUSATTEMPTSTOEXPRESSBASIINPPASTORISBNSAGERETAL,2003,VARIOUSCONDITIONSHAVEBEENOPTIMIZEDINTHISSTUDY,INCLUDINGSIGNALSEQUENCE,CODONUSAGE,TRANSFORMATIONTECHNIQUEANDGROWTHCONDITIONSBACKCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE2MATERIALSANDMETHODS21STRAINS,CULTUREMEDIAANDGROWTHCONDITIONS1PPASTORISINVITROGEN,USAGS115WASUSEDFORHETEROLOGOUSEXPRESSIONOFBASI2YPDMEDIUM1W/VYEASTEXTRACT,2W/VPEPTONE,2W/VDEXTROSEYNBYEASTNITROGENBASEWITHAMMONIUMSULPHATEWITHOUTAMINOACIDSBMSYMEDIACOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE2MATERIALSANDMETHODS3TOINDUCEEXPRESSIONPPASTORISWASGROWNIN500MLBMSYMEDIAAT28C,280RPMAFTER24HGROWTH,EXPRESSIONWASINDUCEDBYADDING50METHANOLTOACONCENTRATIONOF05V/VTOMAINTAININDUCTION50METHANOLWASADDEDTO05V/VEVERY24HCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE2MATERIALSANDMETHODS22CLONING1ANEWPOLYLINKERWASINTRODUCEDINTOPPIC9KBYREPLACINGTHEBAMHINOTIFRAGMENT,INCLUDINGTHEALPHAMATINGFACTORSECRETIONSIGNAL,CREATINGPPIC9KPFIG1COPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE2MATERIALSANDMETHODS23PREPARATIONOFCOMPETENTCELLS1CONDUCTTHESTEPOFTHISEXPERIMENTACCORDINGTOTHEPROCEDUREOFTHISPAPER2THECOMPETENTCELLSWEREEITHERUSEDRIGHTAWAY,ORSTOREDAT80CFORLATERUSECOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE2MATERIALSANDMETHODS24TRANSFORMATION1PRIORTOTRANSFORMATIONALLPLASMIDSWERELINEARIZEDBYBGLIIDIGESTION,FOLLOWINGGELPURIFICATIONUSINGMINELUTEQIAGENHILDEN,GERMANY2TRANSFORMATIONSWEREDONEBYELECTROPORATIONUSINGAGENEPULSERBIORAD,USA,VOLTAGE1500V,CAPACITANCE25FANDRESISTANCE200COPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE2MATERIALSANDMETHODS25SCREENFORSINGLECOPYTRANSFORMANTS1SINGLECOLONIESWEREPICKEDFROMTHEINITIALYNBPLATESANDRESUSPENDEDIN10LWATER2CELLSWERELYSEDBYMICROWAVINGFOR1MINCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE2MATERIALSANDMETHODS26INSOLUBLEBLUESTARCHASSAY1AMY2375NMWASPREINCUBATEDWITHBASIATVARIOUSCONCENTRATIONSRANGINGFROM0TO50NMINATOTALVOLUMEOF400LFOR10MINAT37CINREACTIONBUFFERCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE2MATERIALSANDMETHODS27SOUTHERNBLOTTING1TOTALDNAWASPURIFIEDBYUSINGTHEMASTERPURETMYEASTDNAPURIFICATIONKITFROMEPICENTREMADISON,USA2THEQUALITYANDCONCENTRATIONOFTHEDNAWASDETERMINEDBYAGAROSEGELELECTROPHORESISANDSPECTROPHOTOMETRYCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE2MATERIALSANDMETHODS28PROTEINPURIFICATION1BASIWASPRECIPITATEDFROMTHESUPERNATANTBYADDINGSOLIDAMMONIUMSULPHATENH42SO4TO32MFINALCONCENTRATION,ANDTHESOLUTIONWASSTIRREDFOR30MIN2BASICONTAININGFRACTIONSFROMTHECOLUMNWEREANALYSEDBYSDSPAGEANDPUREFRACTIONSWEREPOOLEDASTHEPRODUCTOFTHEPURIFICATIONBACKCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE3RESULTS31DESIGNOFCODONOPTIMIZEDGENES1TOINVESTIGATEWHETHERALTERATIONOFTHECODONUSAGEINTHEBASICDRTWOCODONOPTIMIZEDCODINGREGIONS,COULDRESULTINHIGHEREXPRESSIONLEVELS,TWOALTERNATIVEBASIGENESWEREDESIGNEDONEFULLYCODONOPTIMIZEDARBASIANDONEWHEREONLYARGININECODONSWEREALTEREDRBASICOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEOFLIFESCIENCE3RESULTS32CLONINGOFBASI,ARBASIANDRBASI1THEORIGINALBASISIGNALPEPTIDEWASREPLACEDBYATRUNCATEDVERSIONOFTHEALPHAMATINGFACTORSECRETIONSIGNALFROMSCEREVISIAE,INORDERTOENSURESECRETIONANDAUTHENTICITY2TOENHANCETHEINITIALBINDINGOFTHESMALLSUBUNITOFTHERIBOSOMETOTHEMRNATEMPLATE,THECONSENSUSKOZAKSEQUENCEKOZAK,1987ACCWASINSERTEDJUSTUPSTREAMOFTHESTARTCODONINALLCONSTRUCTSCOPYRIGHT2014XIANGRUCHEN,ALLRIGHTSRESERVEDCOLLEGEO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