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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Expression of Sea Anemone Equistatin in Potato. Effects of Plant Proteases on Heterologous Protein Production¹

Plant Research International, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands (N.S.O., M.A.J.); and Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia (B.R., B.S.)

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 Desirée) 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.

² These authors equally contributed to the paper.

^* Corresponding author; e-mail maarten.jongsma{at}wur.nl; fax 31-317-418094.

Received November 7, 2002; returned for revision January 12, 2003; accepted May 14, 2003.

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