Iqondo le-protein yokuhlanzeka lifunekayo kuxhomekeke ekusebenziseni ekupheleni kweprotheni.
Kwezinye iiscelo, ukukhishwa okungaqhelekanga kwanele. Nangona kunjalo, ngezinye iindlela, ezifana nokutya kunye namayeza, iqondo eliphezulu lokuhlanzeka liyadingeka. Ukufezekiswa kwezi ndlela ezininzi zokuhlanjululwa kwamaprotheni zisetyenziswe ngokubanzi, kwinqanaba leempawu zokuhlanjululwa.
Isinyathelo ngasinye sokuhlanjululwa kwesiprogram sivame ukuphumela kwinqanaba elithile lokulahleka kwemveliso. Ngoko ke, icebo elifanelekileyo lokucoca iprotheni linye apho izinga eliphezulu lokuhlanjululwa lifikelelwa kumanyathelo ambalwa. Ukukhethwa kwamanyathelo okusetyenziswa kukuxhomekeke kubukhulu, inkokhelo, ubunqamlezo kunye nezinye iipropethi zeeprotheyini ezijoliswe kuyo. Amasu alandelayo alungele ukuhlanjululwa kwiprotheni enye ye-cytosolic. Ukuhlanjululwa kweprotoyinti yeprototic complexes kuyinkimbinkimbi kwaye ifuna ukuba iindlela ezahlukeneyo zisetyenziswe.
Amanyathelo okuQala ukuPhenyiswa kwamaprotheni
Isinyathelo sokuqala ekuhlanjululeni i- intracellular (ngaphakathi kweseli) iiprotheni yilungiselelo lokukhishwa koluhlaza .
I-extract iya kubandakanya umxube odibeneyo wee-proteins ezivela kwi-cytoplasm yeseli, kunye nezinye i-macromolecules ezongezelelweyo, ama-cofactors kunye nezondlo. Ukukhishwa okungekho nto ingasetyenziselwa ezinye izicelo kwi-biotechnology, nangona kunjalo, ukuba ukuhlanjululwa ngumbandela, amanyathelo okuhlanjululwa alandelayo kufuneka alandelwe.
Izicatshulwa zamaprotheni ezingcolileyo zilungiswe ngokususwa kweengxube zamaselula eziveliswa yi-cell lysis, ephunyeziweyo usebenzisa iikhemikhali kunye ne- enzyme , u-sonication okanye i-Press Press. I-debris isuswa ngu-centrifugation, kwaye i-supernatant ifunyenwe. Ukulungiselela amalungiselelo angaphantsi kwama-proteins extracellular angafunyanwa ngokususa nje iiseli ngo-centrifugation.
Kwiimfuno ezithile ze- biotechnology , kukho imfuno ye- enzyme eziphepheleyo: ii-enzyme ezinokunyamezela ukushisa okuphezulu ngaphandle kokuphazamiseka, kwaye ngelixa zigcina umsebenzi ophezulu. Iimpawu ezizivelisa ngamanye amaxesha zibizwa ngokuthi i-extremophiles. Indlela elula yokuhlanjululwa kwiprotheni engaxhatshazelanga ukushisa ukuguqulwa kwamanye amaprotheni kumxube ngokufudumeza, kwaye ukupholisa isisombululo (oko kukuvumela ukuba i-enzyme ephosakeleyo iguquke okanye ibuye ibuye ibuyele kwakhona, ukuba kuyimfuneko.) Iiproteti ezidityanisiweyo ziyakususwa ngo-centrifugation.
Amanyathelo okuhlambulula aphakathi
Kwixesha elidlulileyo, isinyathelo sesibini esifanayo sokuhlanjululwa kwiprotheyini evela kwi-extract crude yenziwa yimpompo kwisisombululo ngamandla amakhulu ase-osmotic (oko kukuthi izisombululo zetyuwa). I-nucleic acids ekukhutshweni okungekho nto ingasuswa ngokutshintshela ama-aggregates akhiwe nge-streptomycin sulphate okanye i-protamine sulfate.
I-protein precipitation idla ngokusetyenziswa nge-ammonium sulfate njengetyuwa.
Amaprotheni ahlukeneyo aya kunqumla kwiindawo ezahlukeneyo ze- ammonium sulfate . Ngokuqhelekileyo, iiprotheyini zesisombululo esiphezulu se-molecular molecule zinyuka kwiindawo ezincinci ze-ammonium sulfate. I-precipitation yetyuwa ayidla ngokukhokelela kwiprotheni ehlambulukileyo kodwa inokunceda ekupheliseni ezinye iiprotheni ezingafuneki kumxube kwaye zijolise kwisampuli. Isalathisi kwisisombululo siya kususwa yi-dialysis nge-tuberous cellulose, i-filtration, okanye i-gel chromatography.
Iiprotokto zamanje ze-biotech zihlala zixhamla kwiikiti ezininzi ezithengiswayo ezinikezela izisombululo ezenziwe ngokuzenzekelayo kwiinkqubo eziqhelekileyo. Ukuhlanjululwa kwamaprotheni kudla ngokusetyenziswa ngokusebenzisa iifayile kunye neentlanzi zokucoca i-gel. Konke omele ukwenze kukulandela imiyalelo kwaye wongeza umthamo ochanekileyo kwisisombululo esifanelekileyo kwaye ulinde ubude bexesha elithile ngexesha uqokelela ukungafihli (oko kuphuma kwenye indawo ekupheleni kwikholam) kwi-tube entsha yokuhlola.
- Izindlela ze-Chromatographic zingasetyenziswa ngokusebenzisa iikholum-top columns okanye i-automated HPLC izixhobo. Ukwahlula ngo-HPLC kunokwenziwa ngoluhlu lwe-reverse-phase, i-exchange exchange okanye i-size-excluding methods, kunye neesampuli ezifunyenwe yi-diode array okanye i-laser technology. A
Iprotheni Ukubonakalisa nokuVavanywa koCoceko
- I-Chromatography yesigaba esichaphazelekayo (RPC) ihlula amaprotheni asekelwe kwi- hydrophobicity yazo . Le ndlela ithetha kakhulu kodwa ifuna ukusebenzisa i-solvents. Ezinye iiprotheni zichithwe ngokungqalileyo nge-solvents kwaye ziya kulahla ukusebenza ngexesha le-RPC. Ngako oko le ndlela ayikhuthazwa kuzo zonke izicelo, ngokukodwa ukuba kuyimfuneko kwiprotheni ekujoliswe kuyo ukugcina umsebenzi.
- I-chromatographic exchange ye-Ion ibhekisela kulwahlulo lweeprotheni ezisekelwe kwintlawulo . Iikholomu zinokulungiswa ukutshintshiselana kwe-anion okanye utshintshiselwano lwentambo. Amanqaku okutshintshela ama-Anion aqukethe isigaba esisigxina kunye nentlawulo ekhangayo ekhangisa iiprotheni ezingekho phantsi. Amakholomu okutshintshwa kweCation ayimilinganiselo echaseneyo, engathandabuzekiyo ekhangayo iiprotheni ezinomtsalane. I-elution yeprotheyini ekujoliswe kuyo iyenziwa ngokutshintsha i-pH kwikholam, ekhokelela ekutshintsheni okanye ekungahambisani neqela lamaqela asebenzayo eprotheyini nganye.
- Ukuchithwa kwe-chromatography ( ukuhlutha i-gel ) ukuhlukanisa ukukhulula ukuhlula iiprotheni ezinkulu ukusuka kwiincinci kuba iinqununu ezinkulu zihamba ngokukhawuleza nge-polymer ehlangeneyo ekhompyutheni. Iiprotheni ezinkulu azifanelanga kwiipolisi kanti iiprotheni ezincinci zenza, kwaye zithatha ixesha elide ukuba zihambe ngekholam ye-chromatography, ngeendlela ezingekho ngqo. I-Eluate iqokelelwa kwisiqulatho seetyhubhu ezahlula iiprotheni ezisekelwe kwixesha lo-elution. Ukucoca i-Gel iyithuluzi eluncedo lokugxininisa isampuli yeprotheni ukususela kwiprotheni ekujoliswe kuyo eqokelelwa kumqulu omncinci we-elution kunokuba uqale wongezwa kwikholam. Amasu okucoca afana nawo angasetyenziswa ngexesha lokuveliswa kweprotheni ephezulu ngenxa yeendleko zabo.
- Ubudlelwane obuchithwa ngumbono luyindlela ekhethekileyo yoku "ukupholisa" okanye ukugqiba inkqubo yokucoca amaprotheni. Ubuninzi kwikholamatrafowuni ikholwaniswe kwii ligands ezibophe ngqo kwiproteinyini ekujoliswe kuyo. Iprotheni iyasuswa kwikholam ngokuhlanjululwa ngesisombululo esinesigxina samahhala. Le ndlela inika iziphumo ezizimeleyo kunye nomsebenzi othile othe xa uthelekiswa namanye amacandelo.
- I-SDS-IPHEPHA i-polyacrylamide gel electrophoresis, eyenziwa phambi kwe-SDS (i-sodium dodecyl sulfate) ebopha kwiiprotheni ezinika inkokhelo enkulu enomnatha. Ekubeni iindleko zazo zonke iiprotheni zilingana ngokulinganayo, le ndlela iwahlukanisa phantse ngokusekelwe kwisayizi. I-SDS-IPHEPHA isoloko isetyenziselwa ukuvavanya ukucoceka kweprotheni emva kwesinyathelo ngasinye kwichungechunge. Njengoko amaprotheni angafunekiyo athatyathwa kancane kumxube, inani lamagqabi abonakaliswe kwi-SDS-PAGE gel liyancitshiswa, de kubekho enye ibhodi emele iprotheni efunwayo.
- I-Immunoblotting yindlela yokubonakalisa iprotheni esebenzayo ngokudibanisa ne-affinity chromatography. Ama-antibodies kwiprotheyini ethile isetyenziswe njengegigesi kwikholomu ye-chromatography. Iprotheni ekujoliswe kuyo igcinwa kwikholam, ize isuswe ngokuhlambulula ikholomu ngesisombululo setyuwa okanye ezinye i-agent. Ama-antibodies axhunyiwe kwiibhalana zokuncedisa i-radioactive okanye idayi ekunokufunyanwa kwiprotheyini ekujoliswe kuyo xa ihlukaniswe kuwo wonke umxube.
Imithombo:
Zubay G. 1988. I-Biochemistry, Edition 2. Macmillan Publishing Co., eNew York, NY, eU.SA.
Amersham Pharmacia Biotech. Ngo-1999. I-Protein Purification Handbook, Edition AB. Amersham Pharmacia Biotech Inc. eNew Jersey, eU.SA. http://www.biochem.uiowa.edu/donelson/Database%20items/protein_purification_handbook.pdf.