A. gossypii transformants were grown on AFM containing 200 μM G418 at 28°C for 7 days. Protein expression was verified by harvesting the mycelium and preparing whole-cell protein extracts of the A. gossypii transformant colonies, as described previously (28 (link)). The prepared extracts were analyzed by immunoblotting using specific antisera (1:2,500 dilutions) for detection of the various FBDPs, except for the extracts containing tamavidin 1, where the blotted extract proteins were probed for bound biotin using the Vectastain ABC alkaline phosphatase system (Vector Laboratories) in combination with a 1-Step NBT/BCIP (nitro-blue tetrazolium/5-bromo-4-chloro-3′-indolyl phosphate) solution (Thermo Scientific). Antisera against Coprinopsis cinerea galectin 2 (CGL2) and Coprinopsis cinerea lectin 2 (CCL2) were described previously (18 (link), 28 (link)). The rabbit antisera against Marasmius oreades agglutinin (MOA), Aleuria aurantia lectin (AAL) and Sordaria macrospora transcript associated with perithecial development 1 (TAP1) were raised against the purified recombinant proteins by Pineda Antikörper-Service (Berlin, Germany). Expression and purification of these proteins from E. coli were carried out as previously described (28 (link), 35 (link), 55 (link)).
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