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. 2000 Feb 15;97(4):1914-9.
doi: 10.1073/pnas.030527497.

The Production of Recombinant Proteins in Transgenic Barley Grains

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Free PMC article

The Production of Recombinant Proteins in Transgenic Barley Grains

H Horvath et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1
The developing endosperm cell of barley. C, chromatin; E, ER; G, Golgi apparatus; H, hordein in protein body; M, mitochondrion; P, plasmodesma; S, starch grain; V, vacuole. (Reproduced with permission from the Carlsberg Foundation.)
Figure 2
Figure 2
Agrobacterium vector pJH271 for expression of the protein-engineered heat-stable (, –1, 4)-β-glucanase during development of the barley endosperm and its deposition in the protein bodies. The β-glucanase gene is codon optimized to a G+C content of 63%. The gene for the synthetic green fluorescent protein (sGFP) is under the control of the 35S promoter, and the bar gene is driven by the maize ubiquitin (pUbi1) promoter and the gene's first intron.
Figure 3
Figure 3
Zymogram assay for identification of homozygous and heterozygous transgenic plants producing heat-stable β-glucanase. Germinated and heated half grains are placed on lichenan-containing agar plates. Congo red binds to lichenan but not to its depolymerized saccharides. (a) Germlings of a homozygous transgenic plant, (b) of a heterozygous transgenic plant, and (c) of a nontransgenic segregant in the F2 of the cross transgenic line 5607 × Ca 803111(ant-499(Apex) × Alexis). An untransformed germling is placed at the top of each plate.
Figure 4
Figure 4
Expression of recombinant heat-stable (, –1, 4)-β-glucanase under the control of the D hordein gene (Hor3) promoter in individual T1 grains. For the codon optimized gene, two individual grains were analyzed, and values are given. The codon optimized gene and a signal peptide for targeting the recombinant protein into endosperm storage protein vacuoles are required for optimal production.
Figure 5
Figure 5
Expression of recombinant heat-stable (, –1, 4)-β-glucanase under the control of a barley high pI α-amylase gene promoter in nine different transgenic lines grown in the field in 3 successive years. GP, Golden Promise.
Figure 6
Figure 6
Identification of heat-stable (, –1, 4)-β-glucanase synthesized with the D hordein gene promoter in maturing grains and with an α-amylase promoter in germinating grains. GP, extract of untransformed Golden Promise; M, molecular marker; P, purified unglycosylated enzyme synthesized in E. coli; Mr, 24 kDa. (A) SDS/PAGE and Coomassie blue staining; (B) Western blot decorated with specific antibody and visualized by chemiluminescence. Lanes: 1, extract of germinating grains expressing the enzyme with the α-amylase promoter; 2–4, extracts of germinating grain containing the recombinant enzyme produced with the Hor3 promoter during grain maturation. The larger apparent molecular mass Mr 28 = kDa) of the barley-produced enzyme is because of N-glycosylation. (C—F) Comparison of the recombinant heat-stable (, –1, 4)-β-glucanase synthesized with the D hordein gene promoter in developing grains with or without the signal peptide. (C and E): SDS/PAGE/Coomassie blue staining; and (D and F) Western blot of extracts containing recombinant enzyme synthesized in transformants with signal peptide (E and F, lanes 1–6) and in transformants without it (C and D, lanes 1–4). The recombinant enzyme synthesized without the signal peptide has an apparent molecular weight characteristic of the unglycosylated protein.
Figure 7
Figure 7
Southern blot analysis of T1 seedlings transgenic for the heat-stable (, –1, 4)-β-glucanase (lanes 5–9 and 10–14). The transgenic plants were obtained by Agrobacterium-mediated transformation by using vector pJH271 with the transgene driven by the Hor3 promoter and translation effected on the ER with the D hordein signal peptide. Ten micrograms of genomic DNA was digested with EcoRI and probed with the digoxigenin-labeled coding region of the heat-stable (, –1, 4)-β-glucanase (620 bp). Lanes: 1, 2, 3 = 20, 4, and 2 copies of the plasmid cut with EcoRI (10.2 kb); 4, Golden Promise; M, molecular marker.

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