CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain
The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of u...
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Frontiers Media S.A.
2021-11-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2021.755788/full |
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| author | Michael Panting Inger Baeksted Holme Jón Már Björnsson Yingxin Zhong Henrik Brinch-Pedersen |
| author_facet | Michael Panting Inger Baeksted Holme Jón Már Björnsson Yingxin Zhong Henrik Brinch-Pedersen |
| author_sort | Michael Panting |
| collection | DOAJ |
| description | The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation. |
| first_indexed | 2024-12-14T06:43:00Z |
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| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-14T06:43:00Z |
| publishDate | 2021-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-cc0f7f46dfa749dd939fde42e1ceeb9d2022-12-21T23:13:09ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2021-11-011210.3389/fpls.2021.755788755788CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley GrainMichael Panting0Inger Baeksted Holme1Jón Már Björnsson2Yingxin Zhong3Henrik Brinch-Pedersen4Department of Agroecology, Research Center Flakkebjerg, Aarhus University, Slagelse, DenmarkDepartment of Agroecology, Research Center Flakkebjerg, Aarhus University, Slagelse, DenmarkORF Genetics, Kópavogur, IcelandNational Technique Innovation Center for Regional Wheat Production, Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of Agriculture, National Engineering and Technology Center for Information Agriculture, Nanjing Agricultural University, Nanjing, ChinaDepartment of Agroecology, Research Center Flakkebjerg, Aarhus University, Slagelse, DenmarkThe use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation.https://www.frontiersin.org/articles/10.3389/fpls.2021.755788/fullbarleyunfolded protein responseheterologous expressionrecombinant proteinCRISPR |
| spellingShingle | Michael Panting Inger Baeksted Holme Jón Már Björnsson Yingxin Zhong Henrik Brinch-Pedersen CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain Frontiers in Plant Science barley unfolded protein response heterologous expression recombinant protein CRISPR |
| title | CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain |
| title_full | CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain |
| title_fullStr | CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain |
| title_full_unstemmed | CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain |
| title_short | CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain |
| title_sort | crispr cas9 and transgene verification of gene involvement in unfolded protein response and recombinant protein production in barley grain |
| topic | barley unfolded protein response heterologous expression recombinant protein CRISPR |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2021.755788/full |
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