鼠粒细胞巨噬细胞集落刺激因子的纯化和复性研究.pdf

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1、药物生物技术386PharmaceuticalBiotechnology2013，20(5)：386～391PurificationandRenaturationofE．coli．DerivedMouseGranulocyte-MacrophageColonyStimulatingFactorCAORong．yue，CHENMeng，GEChi—yu，TANGXiao—tian，LIFei，ZHANGPeng，XUMao—lei，lLIUJing-jingf《1．MinigenePharmacyLaboratory,Sch

2、oolofLifeScienceandTechnology,ChinaPharmaceuticalUniversity，Nan—jg210009China；2．XintrumPharmaceuticalsLtd,N锄ng210017,China)AbstractGranulocytemacrophage—colonystimulatingfactor(GM·CSF)isahematopoieticgrowthfactorandaproinflammatorycyt0kine．RecombinantmouseGM．CSFfr

3、mGM—CSF)isnecessaryinantineoplasticandanti—inflammatoryresearch．ToobtainrmGM—CSF．theencodingregionformGM．CSFwasclonedbetweenNcoIand口0mHIinpET28a．TheproteinwasexpressedintheformofinclusionbodiesinEc0lihoststrainBL21fDE3)．Theexpressionlevelwasmorethan51．6％oftotalcel

4、llysate．Inclusionbodieswerewashedunderoptimizedconditions，anddissolvedinabufercontaining8mol／Lurea．Dilutionmethodcombinedwithpolyethy—leneglyeolfPEG1asaco．solventwaSemployedtorefoldproteinsin2mol／Lurea．Afterion—exchangechromatography，rmGM—CSFwithahighDuritywasobta

5、ined．PurifiedrmGM—CSFhasagoodfidelity，whichmakesitpossibletopromoteproliferationofmyeloidce11sinitroandtoincreasethenumberofleukocyteandneutrophilinvivo．Ahighyieldof8．06mgrmGM—CSFperlitreofcellculturewasachievedbyusingthismethod．Owingtoitshighyieldandsimpleoperati

6、on，thisprotocolissuitableforlarge—scaleproduction·KeywordsRecombinantmousegranulocyte-macrophagecolony—stimulatingfactor，Inclusionbodies，Polyethyleneglycol，Purifi—cation，Refolding，BioactivityCLCNumberQ813DocumentcodeAArticleID1005—8915(2013)05—0386—06agreatdealofi

7、solationtrials_9jGranulocytemacrophage-colonystimulatingfactor(GM—．whichrevealedconundrumofCSF)wasoriginallydugoutasapotentialofgeneratingbothhighcostsandlowyield，thoughglycosylatedproteinwasgranulocyteandmacrophagecoloniesfromhematopoieticcellsobtained．However，th

8、ebiologicalactivityofmouseGM—CSFinmousebonemarrow”．However．aseriesoffunctionsweredoesnotdependonglycosylationstiesbutcorrectsconforma—discoveredsubseque