Akademik Veri Yönetim Sistemi
Tezin Türü: Yüksek Lisans
Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Türkiye
Tezin Onay Tarihi: 2005
Tezin Dili: İngilizce
Öğrenci: Burçak Gençer
Danışman: ZÜMRÜT BEGÜM ÖGEL
Açık Arşiv Koleksiyonu: AVESİS Açık Erişim Koleksiyonu
Özet:In this study, self-catalytic maturation of heterologously expressed pro-galactose oxidase was analysed in E.coli by altering some amino acids which were supposed to play a crucial role in pro-peptide removal. Galactose oxidase (GOase; EC 1.1.3.9) from Fusarium graminearum; having a molecular mass of 68kDa, is a monomeric, copper containing enzyme with an unusual thioether bond. The enzyme is produced as a precursor with an additional 8 amino acid pre- and a 17- amino acid pro-sequence at the N terminus. Previous work has shown that the pre-peptide is removed possibly by a protease during secretion, whereas the 17 amino acid pro-peptide is removed autocatalytically by the aerobic addition of Cu2+ to the precursor, preceding the formation of the thioether bond at the active site. The pro-gao gene was on ProGON1 and ProGOMN1 constructs which were previously established on pET101/D/lacZ vector in England by directed evolution. ProGON1 contains silent mutations at the N-terminus different from native galactose oxidase whereas ProGOMN1 has six further mutations within the mature enzyme, providing high expression. The cleavage site mutations R-1P/A1P, R-1X/A1X, S2A, and the H522A mutation just against the cleavage site in the three dimensional configuration, were carried out by site-directed mutagenesis. Those and some extra mutations were confirmed by DNA sequence analysis. Next, mutant galactose oxidases were expressed in E. coli BL21 Star (DE3), and were purified by Strep-Tactin® Sepharose® column, operating on the basis of affinity chromatography. Subsequently, SDS-PAGE was performed to analyze self-processing by detecting molecular mass difference of protein bands resulting from pro-sequence removal or existence. When the bands obtained in SDS-PAGE were compared, it was seen that the products of original recombinant plasmids, i.e. ProGON1, ProGOMN1; and the mutational