親和層析樹(shù)脂Ni-NTAAgarose的制備及其對(duì)基因重組人腦鈉素的純化研究.pdf

1、天津大學(xué)碩士學(xué)位論文親和層析樹(shù)脂Ni-NTAAgarose的制備及其對(duì)基因重組人腦鈉素的純化研究姓名：蔡友慶申請(qǐng)學(xué)位級(jí)別：碩士專(zhuān)業(yè)：生物化工指導(dǎo)教師：甘一如2000.12.1ABSTRACTAnovelaffinityligand，N—f5-amino-Icarboxypentyl)iminodiaceticacid(NTA)，wassynthesizedbythereactionofN-e—benzyloxycabonyl-L—lys

2、inewithbromoaceticacid，andthencoupledwithepoxy—activatedSepharose@CL一6BtoprepareNTAresin，anaffinityadsorbentforimmobilizedmetalionaffinitychromatography(IMAC)Thepurificationof6XHis—taggedrecombinanthumanbrainnatriureticp

3、eptide(His6-BNPlo)wasinvestigatedbyusingtheaffinityresinmadeinthisgroupByusingicebathtwice，thekeyintermediateofNTApreparation，Nf5-benzyloxycarbonyl—amino-l-carboxypentyl)iminodiaceticacidwassuccessfullypreparedwithyieldu

4、pto78％，andpurifyupto90％TheresultwasconfirmedbyRPHPLC，1HN／VlRandFI“瓜AgarosegelwasactivatedinDMSObyrepeatedlyfeedingepichlorohydrin，andtheconcentrationofepoxygroupsinactivatedgelwasincreasedupto90umo垤drainedgelThentheactiv

5、atedgelcoupledwithNTAtoformaffinityresin(117％N，andhence329umo垤immobilizedligand)AftercoordinatedwithNi2，thecoupledgelcontained93umolNi2／mLgelandabindingcapacityof87mg／mLresinfortheproteinHis6一BNPloAllthesepropertieswereq

6、uitesimilartothesameproductavailableinQiagencompanyHowever，theisothermaladsorptioncmveforgelandproteincouldnottitLangmuirisothermcurvebutrectangularisothelmcurve，whichindicatedtherewasaverystronginteractionbetweenNiNTAan

7、dHis6-BNPloTopurifytheHis6-BNPlo，thepHgradientelutionwasfollowedbyimidazolegradientelutioninonesinglestepbatchpurificationThepurityandyieldofthepurifiedproteinwere85％and70％respecitivelyKeywords：Immobilizedmetalionaffinit