Epsilon Archive for Student Projects

Abstract

The protein Loosenin 1 from the wood degrading fungus Bjerkandera adu-sta (BaLOOS1) has been shown to loosen up recalcitrant biomass and en-hance enzymatic saccharification. BaLOOS1 is the only loosenin protein that has been biochemically characterised so far. An earlier attempt to ex-press BaLOOS1 in Pichia pastoris, with its native signal peptide (SP), indi-cated low expression level (T. Haataja and J. Ståhlberg, unpublished).

The purpose of this study was to exchange the native SP of BaLOOS1 in an attempt to obtain higher expression levels. A pGAP P. pastoris expression plasmid was used that contained the gene for BaLOOS1 with an appended C-terminal His tag, and the native SP was exchanged by Gibson assembly followed by transformation into P. pastoris X33-strain. Four SPs were tested. Two SPs were from loosenin homologs in Trichoderma reesei and P. pastoris, respectively, from a previous study. The two other SPs were the α-factor from Saccharomyces cerevisiae and the Epx1-SP from the most abun-dant secretory protein Epx1 in P. pastoris. All the SP constructs were suc-cessfully inserted. The T. reesei SP gave the strongest band of the expected molecular weight on SDS-PAGE gel of the tested SPs indicating a higher expression level than the others. Specific binding to an IMAC column strongly suggests that the expressed protein is the His-tagged BaLOOS1. However, definite confirmation of the identity remains to be done, e.g. by peptide mapping.