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Deletion mutagenesis of fusarium graminearum nrrl 2903 galactose oxidase in escherichia coli to study structure-function relations and for prospective biosensor applications

Kocuklu, Burçak
Galactose oxidase (GAO; EC from Fusarium graminearum; is a 68kDa, monomeric extracellular enzyme having an unusual thioether bond with a single copper ion at its active site. The enzyme is produced as a precursor with a 25 amino-acid leader peptide, consisting of a 17-amino acid pro- and an 8-amino acid putative pre-peptide, in addition to a signal peptide. Based on previous studies, the 17-amino acid pro-peptide is removed autocatalytically by the aerobic addition of Cu2+ to the precursor in vitro, preceding the formation of the thioether bond at the active site. In our previous study, it was discovered that self-processing of the pro-peptide did not take place when GAO was expressed heterologously in E.coli in its pre-pro- precursor form. In this study, the question whether the 8-amino acid pre-peptide is hindering the self- cleavage of heterologously expressed pre-pro-galactose oxidase in E.coli is tried to be answered by deleting the 8 amino-acid pre-peptide via site-directed mutagenesis. pPre-ProGON1 (encoding wild- type GAO together with the leader peptide with N-terminal silent mutations) and pPre-ProGOMN1 (encoding a variant of GAO together with the leader peptide, with silent mutations at the N-terminus different from native gao and six further mutations within the mature enzyme) which were developed by directed evolution, were used as the plasmid constructs in developing deletion mutations in this thesis study. The pre-peptide deletion was carried out by QuikChange® Site-Directed Mutagenesis approach and confirmed by DNA sequencing. The newly generated pProGON1 and pProGOMN1 constructs were expressed in E. coli BL21 Star (DE3) through pET expression system either by IPTG induction or by auto-induction. Following purification trials of mutant galactose oxidases by different affinity chromatography matrices, SDS-PAGE analysis was performed to figure out whether the pre-deleted galactose oxidase variants recover the ability of self-processing. Comparison of the SDS-PAGE bands substantiated that the pro-peptide is still present at the N-terminus. Accordingly; detection of GAO activity and molecular weight derived from SDS-PAGE also indicated that pro-peptide has no detrimental effect on the proper thioether bond formation at the active site.However, specific activities of Pro-GAOs and Pre-Pro-GAOs are c.13 fold and c.1,8 fold lower than MatGOMN6, respectively, indicating pre-peptide effect on active site conformation. In addition, the prevention of the autoprocessing of the pro-peptide is not due to the presence of the pre-peptide at the N-terminus in E.coli. Moreover, this discovery underlines that the self-catalytic maturation of the galactose oxidase may not be only copper and oxygen dependent but any other mechanism present or absent in E.coli such as posttranslational modifications prevents autoprocessing. It also appears from this study that pro-peptide self-cleavage is independent of the primary amino acid sequence of GAO. On the second part of this study, domain deletions of a variant of mature galactose oxidase, namely MatGOMN6 (with silent mutations at the N-terminus different from native gao and six further mutations within the mature enzyme), were aimed for novel biosensor applications. After His-tag had been substituted for Strep-tag successfully on the construct encoding mature GAO (pMatGOMN6), in order to facilitate downstream processes, deletion and expression studies of domain I/II and domain III were carried out on both derivatives as outlined above. Further optimization and purification studies are underway as a part of another project.