β-glukanaz Enziminin Pichia pastoris Ekspresyon Sisteminde Hücre Dışı Üretimi ve Saflaştırılması

Year 2021, , 620 - 630, 31.08.2021

Mert Karaoglan , Fidan Erden Karaoğlan

https://doi.org/10.18185/erzifbed.972627

Cited By: 2

Abstract

Bu çalışmada, Rhizomucor miehei -1,3-1,4-glukanaz geni, Pichia pastoris ekspresyon sisteminde AOX1 promotorunun regülasyonu altında ifade edilmiş ve en yüksek üretim seviyesini sağlayan klon belirlenmiştir. Kodon optimize edilmiş gen, pPICZA ekspresyon vektörüne bağlanmış ve kompetent P. pastoris KM71H hücrelerine aktarılmıştır. Farklı konsantrasyonlarda zeosin içeren plakalardan otuz transformant seçilerek klonlar protein üretimi için test tüplerinde kültüre alınmıştır. Süpernatant örneklerinde glukanaz enzim aktivitesi ölçülmüş ve en yüksek glukanaz aktivitesi gösteren on klon belirlenmiştir. Ayrıca süpernatant örneklerindeki proteinler SDS-PAGE ile analiz edilmiş ve glukanaz enzimi, yaklaşık 34 ve 38 kDa olmak üzere 2 bant halinde gözlenmiştir. En yüksek glukanaz enzim üretimine sahip klon ile 200 mL indüksiyon ortamında (BMMY) 72 saat boyunca protein üretimi gerçekleştirilmiş ve rekombinant glukanaz enzimi Ni-NTA afinite kromatografisi ile saflaştırılmıştır. Saflaştırma işleminden sonra analiz edilen klonun 79,6 mg/L glukanaz üretim seviyesine ulaştığı belirlenmiştir. Saflaştırma işleminin her aşamasından alınan örneklerin SDS-PAGE analizi, süpernatant örneklerinde de gözlenmiş olan iki protein bandının glukanaz enzimini temsil ettiğini göstermiştir.

Keywords

B-glukanaz, pichia pastoris, rhizomucor miehei, protein saflaştırma

Supporting Institution

ERZİNCAN BİNALİ YILDIRIM ÜNİVERSİTESİ

Project Number

FBA-2019-596

Extracellular Production and Purification of the β-glucanase in Pichia pastoris Expression System

Abstract

In this study, Rhizomucor miehei -1,3-1,4-glucanase gene was expressed under the regulation of AOX1 promoter in the Pichia pastoris expression system, and the clone providing the highest production level was determined. The codon-optimized gene was ligated into expression vector pPICZA and transferred into competent P. pastoris KM71H cells. Thirty transformants were selected from plates containing different concentrations of zeocin and cultured in test tubes for protein production. Glucanase enzyme activities in supernatant samples were measured and ten clones showing the highest glucanase activity were determined. The proteins in supernatant samples were also analyzed by SDS-PAGE and the glucanase enzyme was observed in 2 bands, approximately 34 and 38 kDa. Protein production was performed for 72 hours in 200 mL induction medium (BMMY) with the clone providing the highest glucanase enzyme production and the recombinant glucanase enzyme was purified by Ni-NTA affinity chromatography. After purification, it was determined that the analyzed clone reached 796 mg/L glucanase production level. SDS-PAGE analysis of samples from each step of the purification procedure showed that the two protein bands also observed in the supernatant samples represent glucanase enzyme.

Keywords

β-glucanase, Pichia pastoris, Rhizomucor miehei, Protein purification

Project Number

FBA-2019-596

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