The production of recombinant proteins in transgenic barley grains.
The grain of the self-pollinating diploid barley species offers two modes of producing recombinant enzymes or other proteins. One uses the promoters of genes with aleurone-specific expression during germination and the signal peptide code for export of the protein into the endosperm. The other uses promoters of the structural genes for storage proteins deposited in the developing endosperm. Production of a protein-engineered thermotolerant (1, 3-1, 4)-beta-glucanase with the D hordein gene (Hor3-1) promoter during endosperm development was analyzed in transgenic plants with four different constructs. High expression of the enzyme and its activity in the endosperm of the mature grain required codon optimization to a C+G content of 63% and synthesis as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeting into the storage vacuoles. Synthesis of the recombinant enzyme in the aleurone of germinating transgenic grain with an alpha-amylase promoter and the code for the export signal peptide yielded approximately 1 microgram small middle dotmg(-1) soluble protein, whereas 54 microgram small middle dotmg(-1) soluble protein was produced on average in the maturing grain of 10 transgenic lines with the vector containing the gene for the (1, 3-1, 4)-beta-glucanase under the control of the Hor3-1 promoter.
Horvath
H
H
Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164-6420, USA. Getreidemarkt 9, A-1060 Wien, Austria.
Huang
J
J
Wong
O
O
Kohl
E
E
Okita
T
T
Kannangara
C G
CG
von Wettstein
D
D
D015139:Blotting, Southern; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D018525:Germination; D005983:Glutens; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics; D006031:Glycosylation; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012231:Rhizobium / Q000235:genetics; D012639:Seeds / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics
D010940:Plant Proteins; D011994:Recombinant Proteins; D005983:Glutens; D006026:Glycoside Hydrolases; D000516:alpha-Amylases; C028020:licheninase