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 variants showed no difference in band size, all slightly above 70kDa / indicating pro-sequence presence on all constructs. Non-mutants and some of the mutants showed galactose oxidase activity, signifying proper active site construction by thioether bond formation. ProGOMN1 was submitted for N-terminal amino acid sequencing to be able to assert that a size above 70kDa is not solely due to the existence of a 1 kDa Strep-tag II at C-terminus. Sequencing data affirmed the presence of both the pre-peptide and the pro-preptide showing that processing has not occurred at the N-terminus. Accordingly, in this study, it was shown for the first time that the existence of a pre-pro-peptide at the N-terminus of galactose oxidase does not prevent thioether bond formation at the active site. Furthermore, since the pro-peptide is cleaved autocatalytically, the lack of removal of the pre-peptide in E.coli in the presence of Cu 2+ and oxygen is very likely to be the cause of lack of pro-peptide cleavage. In future studies the region corresponding to the pre-peptide will be deleted to prove this hypothesis.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/3/12607081/index.pdf |
| Date | 01 December 2005 |
| Creators | Gencer, Burcak |
| Contributors | Ogel, Zumrut Begum |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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