FIG. 1.

Expression cassettes constructed to study cleavage of Man5A-Xyn11A fusions. The gene fusions were expressed from the cel7A promoter (prom.), and termination of transcription was ensured by using a cel7A terminator (term.) sequence. The man5A signal (ss) and core-hinge sequences (M₁ to G₄₀₆ in pALK945 and pALK948; M₁ to G₄₁₀ in pALK1021 and pALK1022) were used to code for the carriers, the amdS gene was included as a transformation marker, and the cel7A 3′ flanking region, together with the cel7A promoter, was used to target the expression cassette into the cel7A locus by homologous recombination (for a more detailed description, see Materials and Methods). Synthetic linker sequences coding for an additional Arg in pALK945 and a Kex2-like protease cleavage signal, Lys-Arg (as RDKR), in pALK948 and pALK1022 were included to ensure cleavage of the fusion protein. The expression cassette pALK1021 does not contain an additional signal for proteolytic cleavage. The amino acids encoded by the man5A sequence are shown in regular type, those of xyn11A are in italics, and the synthetic amino acids for proteolytic cleavage are in boldface.