Self-assembly of proglycinin and hybrid proglycinin synthesized in vitro from cDNA

• Published in: Proceedings of the National Academy of Sciences - PNAS 1987-08, Vol.84 (16), p.5525-5529
• Main Authors: Dickinson, C.D, Floener, L.A, Lilley, G.G, Nielsen, N.C
• Format: Article
• Language: English
• Subjects: ADN; Biological and medical sciences; Biological Sciences: Applied Biology; BIOSINTESIS; BIOSYNTHESE; BIOSYNTHESIS; Biotechnology; cDNA; Complementary DNA; DNA; Endoplasmic reticulum; EXPERIMENTACION IN VITRO; EXPERIMENTATION IN VITRO; Fundamental and applied biological sciences. Psychology; Gels; Genetic engineering; Genetic technics; GLYCINE MAX; glycinin; glycinin precursors; GRAINE; Hybridity; IN VITRO EXPERIMENTATION; Industrial agriculture; Industrial research; Methods. Procedures. Technologies; Miscellaneous; peptide synthesis; Plasmids; PROTEINAS; PROTEINE; PROTEINS; SEEDS; Self assembly; SEMILLA; Soybeans; storage proteins; Synthetic digonucleotides and genes. Sequencing; Trimers
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.16.5525

An in vitro system was developed that results in the self-assembly of subunit precursors into complexes that resemble those found naturally in the endoplasmic reticulum. Subunits of glycinin, the predominant seed protein of soybeans, were synthesized from modified cDNAs using a combination of the SP6 transcription and the rabbit reticulocyte translation systems. Subunits produced from plasmid constructions that encoded either Gy4 or Gy5 gene products, but modified such that their signal sequences were absent, self-assembled into trimers equivalent in size to those precursors found in the endoplasmic reticulum. In contrast, proteins synthesized in vitro from Gy4 constructs failed to self-assemble when the signal sequence was left intact (e.g., preproglycinin) or when the coding sequence was modified to remove 27 amino acids from an internal hydrophobic region, which is highly conserved among the glycinin subunits. Various hybrid subunits were also produced by trading portions of Gy4 and Gy5 cDNAs and all self-assembled in our system. The in vitro assembly system provides an opportunity to study the self-assembly of precursors and to probe for regions important for assembly. It will also be helpful in attempts to engineer beneficial nutritional changes into this important food protein.