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Brinkenstråhle, Linus, 2018. Signal peptide exchange for higher expression of Bjerkandera adusta Loosenin 1 in P. pastoris. First cycle, G2E. Uppsala: SLU, Department of Molecular Sciences



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in Pichia pastoris, with its native signal peptide (SP), indi-cated low expression level (T. Haataja and J. Ståhlberg, unpublished).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.

Main title:Signal peptide exchange for higher expression of Bjerkandera adusta Loosenin 1 in P. pastoris
Authors:Brinkenstråhle, Linus
Supervisor:Ståhlberg, Jerry and Haataja, Topi
Examiner:Sandgren, Mats
Series:Molecular Sciences
Volume/Sequential designation:2018:13
Year of Publication:2018
Level and depth descriptor:First cycle, G2E
Student's programme affiliation:NK002 Biology with specialisation in Biotechnology - Bachelor's Programme, 180.0hp
Supervising department:(NL, NJ) > Department of Molecular Sciences
Keywords:Loosenin, cellulose, signal peptide, expansin, Pichia pastoris, recombinant expression
Permanent URL:
Subject. Use of subject categories until 2023-04-30.:Chemistry
Deposited On:12 Sep 2018 11:20
Metadata Last Modified:12 Sep 2018 11:20

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