Isolation and heterologous expressi

Isolation and heterologous expression of GalOx-encoding gene
Mycelium of F. sambucinum from a culture grown in liquid medium was harvested and the genomic DNA was isolated. Degenerated primers based on published sequences were used to amplify the gao gene coding for GalOx including its signal sequence. The gene consists of an open reading frame of 2037 bp encoding a polypeptide of 679 amino acids. The sequence (GenBank accession No. KM052576) contains no introns and a 37 amino acid prepro sequence. The similarity to the protein sequences of GalOx from F. graminearum [5] and F. oxysporum [68] are 96% and 81%, respectively. The amino acid sequence derived from the F. sambucinum gao gene was used to generate a three-dimensional homology model based on the published structure of mature GalOx (1gog) from F. graminearum [5] using SWISS-MODEL [61], [62] and [63] ( Fig. 1). The amino acids in the active site ( Fig. 1B) as well as in the second shell surrounding it are completely conserved. The architecture of the substrate-binding pocket as well as the residues responsible for copper binding are also identical. The changes in amino acid sequences are mainly found on the surface of the protein.