Outchkourov Nikolay S, Rogelj Boris, Strukelj Borut, Jongsma Maarten A
Plant Research International, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands.
Plant Physiol. 2003 Sep;133(1):379-90. doi: 10.1104/pp.102.017293.
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 P(1)-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.
植物越来越多地被用作各种异源蛋白的生产平台，但蛋白质的快速周转会严重限制其稳态表达水平。目前对于参与这一过程的特定植物蛋白酶知之甚少。为了获得对科罗拉多马铃薯甲虫（Leptinotarsa decemlineata Say）幼虫具有抗性的马铃薯（Solanum tuberosum cv Desirée）植株，在强的、光诱导型和组成型启动子的控制下表达了蛋白酶抑制剂equistatin，并将其靶向分泌途径，有无内质网滞留信号均可。所有构建体都产生了相似的逐步蛋白质降解模式，这大大降低了植物中活性抑制剂的含量，导致对科罗拉多马铃薯甲虫幼虫的抗性水平不足。通过对降解产物进行亲和纯化和N端测序，确定了在植物中被切割的氨基酸P(1)位点（天冬酰胺[Asn]-13、赖氨酸-56、Asn-82和精氨酸-151）。用合成底物和抑制剂对参与equistatin降解的蛋白酶进行了表征。激肽原结构域3在体外完全抑制了equistatin的降解。结果表明，精氨酸/赖氨酸特异性和豆球蛋白型Asn特异性半胱氨酸蛋白酶严重阻碍了重组equistatin在植物中的功能积累。提出了提高植物中异源蛋白对蛋白酶抗性的一般策略。
