Correct Post‐Translational Modification and Stable Vacuolar Accumulation of Phytohemagglutinin Engineered to Contain Multiple Methionine Residues

Abstract

Most legume seed storage proteins are deficient in sulfur amino acids. In this study, we demonstrate that replacing specific amino acid residues of a seed protein with methionine residues at positions known to be occupied by methionine residues in homologous proteins, is an effective strategy to create methionine-enriched seed proteins. Mutant phytohemagglutinin polypeptides with three or four methionine residues were found to undergo correct post-translational modifications in transformed cultured tobacco cells and to accumulate stably in the protein storage vacuoles of transgenic tobacco seeds.The proteins of legume seeds generally have low amounts of the sulfur amino acids cysteine and methionine causing them to be nutritionally sub-optimal as a feed for monogastric animals. Several strategies are available to overcome this deficiency in sulfur amino acids: (a) expression in the seeds of sulfur-rich proteins such as the Brazil nut 2s storage protein, (b) alteration of amino acid biosynthesis, and (c) modification of existing storage proteins. All three approaches have been attempted and several have yielded encouraging results. The direct approach, i.e. expressing a heterologous methionine-rich protein, resulted in seeds of tobacco and canola that have a 30% increase in their methionine content (Altenbach et al., 1992;Altenbach et al., 1989). A novel indirect approach was recently reported in which a mutant Escherichia coli-derived aspartate kinase gene that encoded an enzyme desensitized to feedback control, was expressed in tobacco seeds. The seeds had higher levels of free threonine and methionine and protein-bound methionine. The modification of an existing seed storage protein, phaseolin, with a 15-residue peptide containing six methionine residues was attempted by Hoffman et al. (1988). Although the protein was synthesized in tobacco seeds it did not accumulate in these seeds. Using immunocytochemistry, Hoffman et al. (1988) were able to detect the protein in the endoplasmic reticulum and Golgi apparatus, but not in the protein storage vacuoles where storage proteins normally accumulate. Subsequently, the three-dimensional structure of phaseolin was elucidated (Lawrence et al., 1990) and it was realized that Correspondence to M. J. Chrispeels,

Improvement of the nutritional quality of legume seed storage proteins by molecular breeding

“…1994). The modified phytohemagglutinin was stably accumulated in vacuoles of the transgenic tobacco seed (Kjemtrup et al 1994). This method will be also useful in the improvement of other seed storage proteins through protein engineering.…”

Enhancement of essential amino acid contents in crops by genetic engineering and protein design

“…To date, experimental results showing the practicality of these suggested modifications have not been published. A similar approach, however, has been successfully used to engineer bean seed phytohaemagglutinin (PHA) to contain an additional three to four methionine residues (Kjemtrup et al ., 1994). In this case, the introduction of an engineered gene for P. vulgaris PHA protein enriched in methionine in transgenic tobacco plants resulted in correct processing and accumulation of the protein in the seed.…”

Expression of cDNA for a bark lectin of Robinia in transgenic tobacco plants

“…This result suggests that the larger protein was converted to the smaller protein in the doublet during the extraction of tissues with PBS. Phytohemagglutinin, a lectin from the seeds of Phaseolus vulgaris, was similarly produced as a doublet in cultured tobacco cells that had been transformed by a gene for the lectin [30]. The mechanism responsible for the production of the doublet is unknown.…”