Expression of sea anemone equistatin in potato: effects of plant proteases on heterologous protein production

N.S. Outchkourov, B. Rogelj, M.A. Jongsma

    Research output: Contribution to journalArticleAcademicpeer-review

    57 Citations (Scopus)

    Abstract

    Plants are increasingly used as production platforms of various heterologous proteins, but rapid protein turnover can seriously limit the steady-state expression level. Little is known about specific plant proteases involved in this process. In an attempt to obtain potato (Solanum tuberosum cv Desir¿(c)e) plants resistant to Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, the protease inhibitor equistatin was expressed under the control of strong, light-inducible and constitutive promoters and was targeted to the secretory pathway with and without endoplasmic reticulum retention signal. All constructs yielded similar stepwise protein degradation patterns, which considerably reduced the amount of active inhibitor in planta and resulted in insufficient levels for resistance against Colorado potato beetle larvae. Affinity purification of the degradation products and N-terminal sequencing allowed the identification of the amino acid P1-positions (asparagine [Asn]-13, lysine-56, Asn-82, and arginine-151) that were cleaved in planta. The proteases involved in the equistatin degradation were characterized with synthetic substrates and inhibitors. Kininogen domain 3 completely inhibited equistatin degradation in vitro. The results indicate that arginine/lysine-specific and legumain-type Asn-specific cysteine proteases seriously impede the functional accumulation of recombinant equistatin in planta. General strategies to improve the resistance to proteases of heterologous proteins in plants are proposed
    Original languageEnglish
    Pages (from-to)379-390
    JournalPlant Physiology
    Volume133
    DOIs
    Publication statusPublished - 2003

    Keywords

    • vacuolar processing enzyme
    • thyroglobulin type-1 domain
    • endoplasmic-reticulum
    • cysteine proteinases
    • monoclonal-antibody
    • transgenic plants
    • asparaginyl endopeptidase
    • mammalian legumain
    • pichia-pastoris
    • actinia-equina

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