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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Autores principales:	Michael Panting, Inger Baeksted Holme, Jón Már Björnsson, Yingxin Zhong, Henrik Brinch-Pedersen
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	barley unfolded protein response heterologous expression recombinant protein CRISPR Plant culture SB1-1110
Acceso en línea:	https://doaj.org/article/cc0f7f46dfa749dd939fde42e1ceeb9d
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spelling	oai:doaj.org-article:cc0f7f46dfa749dd939fde42e1ceeb9d2021-11-15T05:55:37ZCRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain1664-462X10.3389/fpls.2021.755788https://doaj.org/article/cc0f7f46dfa749dd939fde42e1ceeb9d2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fpls.2021.755788/fullhttps://doaj.org/toc/1664-462XThe 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.Michael PantingInger Baeksted HolmeJón Már BjörnssonYingxin ZhongHenrik Brinch-PedersenFrontiers Media S.A.articlebarleyunfolded protein responseheterologous expressionrecombinant proteinCRISPRPlant cultureSB1-1110ENFrontiers in Plant Science, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	barley unfolded protein response heterologous expression recombinant protein CRISPR Plant culture SB1-1110
spellingShingle	barley unfolded protein response heterologous expression recombinant protein CRISPR Plant culture SB1-1110 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
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.
format	article
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
title	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_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_sort	crispr/cas9 and transgene verification of gene involvement in unfolded protein response and recombinant protein production in barley grain
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/cc0f7f46dfa749dd939fde42e1ceeb9d
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_version_	1718428581837668352

CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain

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