Chiyero cheprotein purity chinodaro chinoenderana nechinangwa chekushandiswa kweprotein.
Kune mamwe maitiro, kubudiswa kusina kukwana kwakakwana. Zvisinei, kune zvimwe zvinoshandiswa, zvakadai sezvokudya nemishonga, inzvimbo yakakwirira yekuchena inodiwa. Kuzadzikisa iyi mapuroteni akawanda ekuchenesa nzira dzinowanzoshandiswa, mumutsara wekunatswa matanho.
Purogiramu imwe neimwe yekuchenesa inowanzowedzera mune imwe yero yekurasikirwa kwemagetsi. Nokudaro, yakakodzera mapuroteni kuchenesa nzira ndeye iyo iyo yakakwirira yekunatswa inosvika mumatanho mashomanana. Kusarudzwa kwezvikwiriso zvinoshandiswa zvinoenderana nehukuru, hutengo, solubility nezvimwe zvinhu zvepuroteni inotarisirwa. Maitiro aya anotevera akakodzera pakuchenesa imwe cytosolic protein. Kucheneswa kweyytosolic protein complexes kwakaoma uye inowanzodikanwa kuti nzira dzakasiyana dzinoshandiswa.
Matanho okutanga kwePurotini Kucheneswa
Nhanho yekutanga mukuchenesa intracellular (mukati memusero) mapuroteni ndeyekugadzirira kwekutsvaga kusina kukodzera .
Iyo yakasanganiswa ichava nemusanganiswa wakaoma wepuroteni yese kubva muyero cytoplasm, uye mamwe macromolecules, mamwe majekiti, uye michina. Izvo zvakasvibiswa zvinogona kushandiswa kune zvimwe zvekushandiswa mu biotechnology, zvisinei, kana chichena chiri dambudziko, inotevera kucheneswa matanho inofanira kuteverwa.
Zvinyorwa zvepurotini zvinyorwa zvinogadzirwa nekubviswa kwemasvosve emasero anokonzerwa nesero lysis, iyo inowanikwa achishandisa makemikari uye enzymes , sonication kana French Press. Izvo zvakasvibiswa zvinobviswa ne centrifugation, uye kupona kunowanikwa zvakare. Zvigadzirwa zvisina kukodzera zveprotein extracellular zvinogona kuwanikwa nekungobvisa masero ne centrifugation.
Kune mamwe maitiro ezvigadzirwa zve biotechnology , pane chido chezvinokonzera zvinokonzerwa nemhepo yakasimba: Enzyme dzinogona kubvumira kutonhora kwakanyanya pasina kuchenesa, uye nekuchengetedza basa rakakwirira. Zvisikwa zvinowanzozvibudisa dzimwe nguva zvinonzi zvinopedzisira. Nzira iri nyore yekuchenesa puroteni inopisa inopisa ndeyokureva mamwe mapuroteni mumusanganiswa nekupisa, uye kunotonhorera mhinduro (nokudaro kubvumira kuti enzyme inopisa ishanduke kugadzirisa kana kuti inodedolve, kana zvichidikanwa.) Zviratidzo zvepineti zvinogona kubva zvabviswa ne centrifugation.
Kupatsanurwa kwepakati pezvikamu
Munguva yakapfuura, danho rechipiri rinowanzoshambidza kuchenesa puroteni kubva kunzvimbo yakasvibiswa yaive nekupinza mune imwe sarudzo nepamusoro yeasmotic simba (kureva munyu mhinduro). Nucleic acids mukutorwa kwakasvibiswa kunogona kubviswa nekugadzirisa masangano akaumbwa ne streptomycin sulphate kana protamine sulfate.
Puroteni yakagadziriswa inowanzoshandiswa uchishandisa ammonium sulfate semunyu.
Zviratidzo zvepuroteni zvakasiyana-siyana zvicharamba zvichiita zvakasiyana-siyana zveammonium sulfate . Kazhinji, mapuroteni epamusoro emakemikisi ekuremera anowedzera mumatsetse emadzimai eammonium sulfate. Mvura inonaya haifaniri kutungamirira kupuroteni yakanatswa asi inogona kubatsira mukubvisa mamwe mapuroteni asina kudiwa mune musanganiswa uye kuisa shanduko. Mazamu ari muhutano anobva abviswa ne dialysis kuburikidza ne porous cellulose tubing, kufuta, kana gel kubviswa chromatography.
Mazuva ano zvinyorwa zvebiotech zvinowanzoshandisa mikana yakawanda inotengeswa inowanikwa yakagadzirirwa-yakagadziriswa zvigadziriswa zvinowanikwa. Puroteni kuchenesa kunowanzoitwa kuburikidza nemafuta uye yakagadzirwa gel filtration columns. Zvose zvaunofanirwa kuita ndeyekutevera mirayiridzo uye wobva wawedzera mhando yakarurama yesarudzo yakarurama uye omirira nguva yakareba yakatarwa paunounganidza kurega (izvo zvinobuda kune rumwe rutivi rwekamuri yacho) mune itsva test test.
- Chromatographic nzira dzinogona kushandiswa uchishandisa mabhenji-pamusoro mapuranga kana automator HPLC midziyo. Kuparadzaniswa neHPLC kunogona kuitwa ne-reverse-phase, ion-exchange kana maitiro-kurega kushandiswa, uye sampuli dzinowanikwa nehudhiyasi yakazara kana laser teknolojia. A
Zviratidzo zvePurotini uye Kuongorora kwekucheneswa
- Reverse-phase chromatography (RPC) inoparadzanisa mapuroteni anobva pane hydrophobicity yavo . Nzira iyi inonyanya kusarudzwa asi inoda kushandiswa kwezvinhu zvinokonzerwa nemagetsi. Zvimwe mapuroteni anogadziriswa zvachose ne-solvents uye acharasikirwa nebasa munguva ye RPC. Nokudaro iyi nzira haina kukurudzirwa kune zvose zvishandiso, kunyanya kana zvichidikanwa kuti chirongwa cheprotein chichengetedze kuchengeta basa.
- Ion-kushandiswa kwechromatography inoreva kuparadzaniswa kwemapurotini anobva pamhosva . Columns inogona kunge yakagadzirirwa anion exchange kana cation exchange. Anion exchange exchange columns ine chikamu chakasimudza nemhosva yakanaka inokwezva mapuroteni anokanganisa zvakashata. Cation exchange exchange columns ndiyo inoshandiswa, inoshandiswa zvakashata zvitsva zvinokwezva zvakanaka zvinokonzera mapuroteni. Elution yechirongwa chepuroteni (s) inogadzirwa nekushandura pH mumutsetse, iyo inoguma nekuchinja kana kusagadzikana kwemapoka akashandiswa anotora mapuroteni ega ega.
- Kukura-kurega chromatography ( gel filtration ) inoparadzanisa mapuroteni makuru kubva kune maduku kubva apo makuru makuru anofamba achifamba nokukurumidza kuburikidza ne cross-linked polymer mumutsara we chromatography. Izvo mapuroteni makuru haafaniri kupinda mumapuroresiti epo apo mapurotini maduku akaita, uye anotora nguva yakareba kuti adzoke mumutsara we chromatography, nenzira yavo isinganzwisisiki. Eluate inounganidzwa mune zvakakoswa zvepabhesi zvakaparadzanisa mapuroteni anobva munguva ye elution. Gel filtration chinhu chakakosha chokuisa purogiramu yemapuroteni kubva apo chinangwa cheprotein chinounganidzwa mudiki shoma elution pane yakatanga kuwedzerwa ku column. Saizvozvowo kushandiswa kwemafuta kunogona kushandiswa panguva yakawanda yeproteiniti yekugadzira nekuda kwezvavanoita-zvinobudirira.
- Kubatana chromatography inzira inobatsira ye "kupima" kana kupedzisa mapuroteni kuchenesa. Uwandu mumutsetse we chromatography uri mucheka-wakabatanidzwa kune magirasi anosunga zvakananga kumapuroteni anodiwa. Puroteni yacho inobva yabviswa kubva kune imwe koronho nekuchenesa nechinangwa chine mahinda. Iyi nzira inopa zvikamu zvakakwana uye basa rakanyanya kunyanya kuenzaniswa nezvimwe nzira.
- SDS-PAGE ipolyacrylamide gel electrophoresis, inoitwa pamberi pe SDS (sodium dodecyl sulfate) iyo inosungira mapuroteni achivapa mambure makuru asina kunaka. Sezvo mitoro yeproteine yose yakaenzana zvakaenzana, iyi nzira inovaparadzanisa zvakakwana zvakakwana maererano nehukuru. SDS-PAGE inowanzoshandiswa kuedza kuchena kwemapuroteni mushure mega imwe neimwe muzvikamu. Sezvo mapuroteni asingadiwi anozobviswa zvishoma nezvishoma kubva musanganiswa, nhamba yezvikwata zvakaratidzwa pa SDS-PAGE gel yakaderera, kusvikira pane boka rimwe chete rinomiririra protein.
- Immunoblotting ipurogiramu yekuona maitiro eprotini inoshandiswa pamwe chete nehukama hwemagariro echromatography. Zvirwere zvepuroteni zvepuroteni chaiyo zvinoshandiswa sezvinongoita pamusana wechromatography column. Puroteni inotarirwa inochengeterwa pahomwe yacho, uye inobviswa nekuchenesa iyo koromo nechinangwa chemunyu kana mamwe mashizha. Zvirwere zvakasanganiswa ne-radioactive kana kuti mavara zvinodha rubatsiro pakuonekwa kweproteinini inorondedzerwa kana yakaparadzana kubva kune imwe sanganiswa.
Sources:
Zubay G. 1988. Biochemistry, Edition 2nd. Macmillan Publishing Co., New York, NY, USA.
Amersham Pharmacia Biotech. 1999. Protein Purification Handbook, Edition AB. Amersham Pharmacia Biotech Inc. New Jersey, USA. http://www.biochem.uiowa.edu/donelson/Database%20items/protein_purification_handbook.pdf.