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| Hauptseite > Publikationsdatenbank > Targeted knockout of barley endosperm-specific storage proteins as a prerequisite for molecular farming purposes |
| Hauptseite > Publikationsdatenbank > Targeted knockout of barley endosperm-specific storage proteins as a prerequisite for molecular farming purposes |
| Conference Presentation (After Call) | FZJ-2023-00594 |
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2022
Abstract: The concept for producing valuable proteins in plants is known as molecular farming. Besides transient expression using viral vector systems in tobacco leaves, the cereal grain is a natural bioreactor ideal for storing proteins under ambient conditions. Therefore, the cereal grain provides a cost-effective, easily scalable expression system for producing high-value proteins in the starchy endosperm. CRISPR-mediated targeted knockout of barley Hordein B1 family members was achieved, and Sanger and deep amplicon sequencing genotyped mutant plants. ELISA and Western blotting performed the abundance and quantification of the recombinant protein overexpressed in wildtype and mutant background. An automated phenotyping device evaluated the morphological changes of the mutant grains. To increase protein yield and overcome the competition between endogenous storage protein accumulation and the high-value protein, a targeted knockout of the Hordein B1 family members by using Cas9 technology was performed. The recombinant human Epidermal Groth Factor (EGF) was overexpressed in wildtype and horb1 mutant background as a proof-of-concept. Grains of the resulting horb1 mutants showed altered biometric traits, reduced total protein and hordein content, and delayed germination behaviour compared to wild type segregants. Western blotting and ELISA results confirmed a high EGF accumulation in the horb1 mutant background. In conclusion, the strategy of higher abundance of a recombinant protein in barley grains was successfully demonstrated by reducing endogenous storage protein accumulation. Further fine-tuning is required to minimize the germination disadvantage.
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