Yintoni na indlela yokucoca yeinkqubo yokucoca iiprotheyini? Kuyimfuneko ukwazi ikhowudi ye-DNA yolandelelwano lweprotheyini ecocekileyo, ukubona ukuba zeziphi iiseli okanye izicubu ezichazwe ngokugqithisileyo kwijeni ekujoliswe kuyo, kunye nokuyila iiprimer zegene zokukhulisa i-arf yesiqwenga se-DNA ekujoliswe kuso. Oku kubizwa ngokuba kukufumana amaqhekeza ekujoliswe kuwo.

Ukwakhiwa kwevektha yokubonakalisa: Kwi-prokaryotic okanye i-eukaryotic expression vector eneempawu ze-gene expression ngokwayo, eyona ngxaki iphambili yeli nyathelo kukwakha i-plasmid kunye ne-gene of interest, kunye ne-expression system. Ixesha lokubonisa iprokaryotic lifutshane, ixabiso liphantsi, kwaye inani elikhulu lokubonakaliswa liphambili; i-gene ayichazwanga kwi-E. coli, kwaye ingxaki yenzeka kwi-codon optimization. Ngokuqwalasela umsebenzi ongcono kunye nokuhlanjululwa kweprotheni, abaphandi bakhetha ukuvakalisa kwimvubelo yePichi. Ukubonakaliswa okunempumelelo kokwenza icodon optimization kubalulekile.

Yintoni indlela yokucoca inkqubo yeprotheyini yokucoca:

1. Imvula.

2. I-Electrophoresis: Iprotheyini ehlawuliswayo iphezulu okanye iphantsi kunendawo yayo ye-isoelectric kwaye inokuhanjiswa kwi-electrode engafanelekanga okanye i-electrode efanelekileyo yentsimi yombane kwintsimi yombane. Inkxaso yefilimu ye-electrophoresis, i-electrophoresis, njl.

3. I-Dialysis: Indlela esebenzisa iingxowa ezimbini ze-dialysis ukwahlula iimolekyuli ezinkulu kwiiprotheni kunye ne-molecular organic compounds.

4. I-Chromatography: I-ion exchange chromatography isebenzisa iipropathi zamahhala. Ngaphantsi kwe-pH ethile, iintlawulo kunye neempawu zeeprotheni zihluke, kwaye ziyakwazi ukuhlukana nge-ion exchange chromatography. Kwi-anion exchange chromatography, iiprotheyini ezinamandla aphantsi angalunganga zikhutshwa kuqala. Izisefu zemolekyuli, ezaziwa ngokuba yi-gel filtration. Iiprotheyini ezincinci zingena kwii-pores kwaye zihlala kuzo ixesha elide. Iiprotheyini ezinkulu azikwazi ukungena kwi-pores kwaye ziphume ngokuthe ngqo.

5. Yintoni indlela yokucoca yeinkqubo yokucoca iiprotheyini? I-Ultracentrifugation: Ukucocwa kweeprotheyini kungasetyenziselwa ukumisela ubunzima be-molecular kwaye ingasetyenziselwa njengeprotheni. Ukuqulunqwa kweeprotheni ezinobunzima obuhlukeneyo buhlukaniswe.