56] In vitro processing of plant preproteins

Abstract

This chapter proposes to develop a cell-free system for the translation, processing and packaging of plant preproteins that demonstrates the synthesis, processing, and deposition of zein polypeptides and α-amylase. The method uses a nuclease-treated rough endoplasmic reticulum (RER)-enriched preparation derived from developing maize endosperm in combination with a wheat germ translation system. The procedure involves material required for membrane preparation, isolation of membranes, preparation of treated membranes, and in vitro protein synthesis and processing. Nonpigmented kernels, such as Illinois High Protein, are preferred to colored kernels. In vivo , zein is synthesized in the maize endosperm on polyribosomes of the RER cisternae that subsequently enlarge to form globular protein bodies. The procedures for the isolation and preparation of treated maize RER membranes can probably be readily adapted to other plant species. Before setting up a synthesis and processing reaction, the membranes are treated with micrococcal nuclease to destroy endogenous zein messages.