A modified storage protein is synthesized, processed, and degraded in the seeds of transgenic plants

Abstract

In vitro mutagenesis was used to supplement the sulfur amino acid codon content of a gene encoding β-phaseolin, a Phaseolus vulgaris storage protein. The number of methionine codons in the phaseolin gene was increased from three to nine by insertion of a 45 base pair (bp) synthetic duplex. Either modified or normal phaseolin genes were integrated into the genome of tobacco plants through Agrobacterium tumefaciens-mediated transformation. Although similar levels of phaseolin RNA are detected in seeds of plants transformed with either the normal or modified (himet) gene, the quantity of himet protein is consistently much lower than normal β-phaseolin. Himet phaseolin is expressed in a temporal- and organ-specific fashion, and is N-glycosylated and assembled into trimers in the manner of normal phaseolin. After germination, both types of phaseolin are hydrolyzed, but the himet protein is more quickly degraded. Electron microscopic immunocytochemical observations of developing seeds indicate that the himet protein is primarily localized in the endoplasmic reticulum (ER) and in Golgi apparatus secretion vesicles. Himet phaseolin is absent from protein storage vacuoles, termed protein bodies, where normal phaseolin is deposited in transgenic tobacco. We interpret the immunocytochemical data to indicate that himet phasolin is transported through the ER and Golgi apparatus and is then degraded in Golgi secretion vesicles or the protein bodies.

Zeolin. A New Recombinant Storage Protein Constructed Using Maize γ-Zein and Bean Phaseolin

“…b-Zein is also a component of maize protein bodies (Lending and Larkins, 1989;Mü ntz, 1998). From a production point of view, that experiment failed because the protein was very unstable in storage vacuoles, but scientifically it represented the first important demonstration that even relatively minor sequence modifications can lead to protein instability in transgenic plants (Hoffman et al, 1988). The construct we have produced is much more stable.…”

“…The introduction of a 15-amino acid Met-rich peptide of b-zein into phaseolin was the first effort to improve the nutritional value of a storage protein (Hoffman et al, 1988). b-Zein is also a component of maize protein bodies (Lending and Larkins, 1989;Mü ntz, 1998).…”

Subcellular Targeting and Purification of Recombinant Proteins in Plant Production Systems

“…(Hoffman et al, 1988). Perhaps the most surprising finding is that correct targeting is apparently independent of the size of the C-lerminal tag, which in lhis case is over three tilnes the size of the oleosin.…”

Genetic contributions to agricultural sustainability

“…Early attempts to increase the content of methionine in seeds by transgenic expression of genes for endogenous storage proteins mutated to add extra methionine residues were unsuccessful (e.g. Hoffman et al 1988). A better strategy was the creation of a synthetic gene encoding an artificial protein rich in essential amino acids.…”