Rotavirus VP7 bacterial expression, purification and characterisation

Abstract

Reoviridae is a family of icosahedral viruses such as rotavirus, a virus that infects both humans and animals, resulting in dehydration and diarrhoea. Rotavirus infections cause high mortality rates in infants in low and middle income countries. The virus contains several different structural proteins, with VP7 acting as an outer capsid layer protein. This study aimed to overexpress, purify and characterise rotavirus VP7. VP7 was expressed predominantly in the insoluble fraction of both BL21 (DE3) and C43 (DE3) pLysS bacterial cells regardless of temperature and induction time conditions. Purifying VP7 using ion exchange and affinity (His-Tag) chromatography via immobilised metal affinity chromatography (IMAC) from the soluble fraction was not successful, but purification of solubilised protein from the insoluble fraction resulted in 100% pure VP7 with a single affinity (His Tag) chromatography step. A yield of 0.013 mg VP7 was obtained from a litre of bacterial culture. Purified VP7 showed secondary structural characteristics of the protein. Intrinsic tryptophan fluorescence indicated a tertiary structure of a folded protein, which, on heating, undergoes conformational rearrangements at temperatures ranging from 20 to 90°C but never completely unfolds.

Reoviridae i matsambu wa ndyangu wun'we wa swi tsongwatsongwani w Icosahedral viruses i xitsongwatsongwani xa tlawa wun'we xikan'we na lexi vange i Rotavirus, i swi tsongwatsongwani kumne ti virus leti khomaku kumbe ku tlulela eka vanhu na swiharhi, leswi heteleku switisa ku omeriwa ka mikolo, torha na chuluko. Ntluleto wa Rotavirus swi vanga mafu yale henhla eka vatsongwani kumbe eka vana vatincece eka matiku lawaha hluvukaku na lawa ya hanyaka evuswetini. Xi tsongwatsongwani kumbe virus leyi, yi kumeka yiri na nhlayo yale henhla ya swiga swo hambana hambana swati protein. Yin'wani yatona kunga leyi vuriwaka VP7 leyi tirhaku tani hi xikhamba xa protein. Ntsariso dyondo lowu wuna xikongomelo xo komba ntikelo wa kona, ku basisiwa naku hambana hambana ka Rotavirus VP7. VP7 yi kombisile ntikelo wa yona na matimba yo thlandlukela ehenhla ku tlula swiga swa BL21 (DE3) na C43 (DE3) pLysS bacterial cells handle ka kuhambana ka timheho tama hiselo ya tshamiseko wa mikarhi. Sawutiso kumbe ku basisiwa ka VP7 hiku tirhisa cincaniso wa swihlawulekiso swa ion na ntshembo (His Tag) chromatography kusuka eka swiga hikwaswo leswi nga tirhisiwa naku kutsandeka is swona hi thlelo ra wona, kambe ntirho wo hluta ku mbhasiso nawo nhlantswa awu humelelangi, kambe mbhasiso wa swihlawulekiso wale hehla wa ti protein ku suka eka swiphemu hikwaswo swa swiga swantirho leswi nga hluleka swi tise mbuyelo wo basa hi xiga xa 100% VP7 hi katsakanyo wo twisiseka wa goza ra (His Tag) chromatography. Ku humelela kale hansi ka 0.013 mg hi litara ya ndhavuko wa switsongwatsongwani kumbe leswi vuriwaka bacterial culture ku kumekile naku humela eri valeni. Mbhasiso katsakanyo hika mbirhi wa VP7 wu konbiso mbuyelo wa xivumbeko xaku hambana hambana kati protein. Nkombiso wa mbuyelo wale henhla wa Intrinsic tryptophan fluorescence wu kombise songano wati protein leti laha ku hisaku ti kombisa mbuyelo wa hlanganiso wa timheho ta mahiselo yo suka eka 20 kuya fika eka 90°C kambe swinga vonaki hiku hetiseka kuya emahlweni.

I‐Reoviridae wumndeni wamagciwane e‐icosahedral afana ne‐rotavirus, igciwane elithelela abantu nezilwane okuholela ekubanjweni yisisu. Ukutheleleka nge‐Rotavirus kubangela amazinga aphezulu okufa ezinganeni emazweni asathuthuka. Leli gciwane liqukethe amaprotheni ahlelekile ahlukene, i‐VP7 isebenza njengeprotheyini engqimba yangaphandle ye‐capsid. Lolu cwaningo kuhloswe ngalo ukusebenzisa i‐overexpress, ukuhlanza kanye nokufaka i‐rotavirus VP7. I‐ VP7 ivezwe kakhulu engxenyeni engafakwanga ye‐BL21 (DE3) ne‐C43 (DE3) pLysS amaseli wegciwane ngaphandle kwezimo zokushisa nezikhathi zokungeniswa. Ukuhlanza i‐VP7 kusetshenziswa ukushintshaniswa kwe‐ion nobudlelwano (i‐HisTag) i‐chromatography evela engxenyeni encibilikayo akuphumelelanga kepha ukuhlanjululwa kwamaprotheni ancibilikisiwe aqhamuka engxenyeni engancibiliki kuholele ku‐100% VP7 emsulwa ngesinyathelo esisodwa sokusondelana (HisTag) chromatography. Kutholwe isivuno esiphansi se‐0.013 mg ilitha ngalinye lesiko lamagciwane. I‐VP7 ehlanziwe ikhombise ukwakheka kwesibili kweprotheni. I‐tryptophan fluorescence yangaphakathi ikhombise ukwakheka okuphakeme kweprotheyini eligoqiwe okuthi lapho kufudunyezwa kube nokuhlelwa kabusha okuhambisanayo emazingeni okushisa asukela ku‐20 kuye ku‐90 ° C kepha kungaze kwenzeke ngokuphelele.