11 research outputs found
Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii
To explore the potential of Ashbya gossypii as a
host for the expression of recombinant proteins and to
assess whether protein secretion would be more similar to
the closely related Saccharomyces cerevisiae or to other
filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase
I (CBHI) from the fungus Trichoderma reesei were
successfully expressed in A. gossypii from plasmids
containing the two micron sequences from S. cerevisiae,
under the S. cerevisiae PGK1 promoter. The native signal
sequences of EGI and CBHI were able to direct the
secretion of EGI and CBHI into the culture medium in A.
gossypii. Although CBHI activity was not detected using 4-
methylumbelliferyl-ÎČ-D-lactoside as substrate, the protein
was detected by Western blot using monoclonal antibodies.
EGI activity was detectable, the specific activity being
comparable to that produced by a similar EGI producing S.
cerevisiae construct. More EGI was secreted than CBHI, or
more active protein was produced. Partial characterization
of CBHI and EGI expressed in A. gossypii revealed
overglycosylation when compared with the native T. reesei
proteins, but the glycosylation was less extensive than on
cellulases expressed in S. cerevisiae.Fundação para a CiĂȘncia e a Tecnologia (FCT