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

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 SP...

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Bibliographic Details
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
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Description
Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.84.16.5525