Mutation of cysteine residues increases heterologous expression of peach expansin in the methylotrophic yeast pichia pastoris

Kaori Matsuyama, Naoki Sunagawa, Kiyohiko Igarashi*

*Corresponding author for this work

    Research output: Contribution to journalArticleScientificpeer-review

    4 Citations (Scopus)

    Abstract

    The study of Carbohydrate-Active enZymes (CAZymes) associated with plant cell wall metabolism is important for elucidating the developmental mechanisms of plants and also for the utilization of plants as a biomass resource. The use of recombinant proteins is common in this context, but heterologous expression of plant proteins is particularly difficult, in part because the presence of many cysteine residues promotes denaturation, aggregation and/or protein misfolding. In this study, we evaluated two phenotypes of methylotrophic yeast Pichia pastoris as expression hosts for expansin from peach (Prunus persica (L.) Batsch, PpEXP1), which is one of the most challenging targets for heterologous expression. cDNAs encoding wild-type expansin (PpEXP1_WT) and a mutant in which all cysteine residues were replaced with serine (PpEXP1_CS) were each inserted into expression vectors, and the protein expression levels were compared. The total amount of secreted protein in PpEXP1_WT culture was approximately twice that of PpEXP1_CS. However, the amounts of recombinant expansin were 0.58 and 4.3 mg l−1, corresponding to 0.18% and 2.37% of total expressed protein, respectively. This 13-fold increase in production of the mutant in P. pastoris indicates that the replacement of cysteine residues stabilizes recombinant PpEXP1.

    Original languageEnglish
    Pages (from-to)397-403
    JournalPlant Biotechnology
    Volume37
    Issue number4
    DOIs
    Publication statusPublished - 2020
    MoE publication typeA1 Journal article-refereed

    Keywords

    • CAZymes
    • Cysteine
    • Expansin
    • Heterologous expression
    • Pichia pastoris

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