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Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant
Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endo...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035899/ https://www.ncbi.nlm.nih.gov/pubmed/33868796 http://dx.doi.org/10.7717/peerj.10851 |
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author | Kim, Kibum Kang, Yang Joo Park, Se Ra Kim, Do-Sun Lee, Seung-Won Ko, Kinarm Ponndorf, Daniel Ko, Kisung |
author_facet | Kim, Kibum Kang, Yang Joo Park, Se Ra Kim, Do-Sun Lee, Seung-Won Ko, Kinarm Ponndorf, Daniel Ko, Kisung |
author_sort | Kim, Kibum |
collection | PubMed |
description | Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endoplasmic reticulum retention motif (GA733-FcK) into the Cowpea mosaic virus (CPMV)-based transient plant expression vector, pEAQ-HT. Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ-HT-GA733-Fc and pEAQ-HT-GA733-FcK was infiltrated into the leaves of Nicotiana benthamiana plants. To optimize harvesting of leaf to express therapeutic glycoproteins both spatially and temporally, protein expression levels at various leaf positions (top, middle, and base) and days post-infiltration (dpi) were investigated. The GA733-Fc and GA733-FcK genes were detected in leaves at 1–10 dpi using PCR. As assessed by western blot, GA733-Fc and GA733-FcK were expressed at the highest levels in the top leaf position at 5 dpi, and GA733-FcK was expressed more than GA733-Fc. The proteins were successfully purified from infiltrated N. benthamiana leaves using protein A affinity chromatography. ELISA verified that an anti-GA733 antibody recognized both purified proteins. Thus, a functional GA733-Fc colorectal cancer vaccine protein can be transiently expressed using a CPMV virus-based vector, with an optimized expression time and leaf position post-infiltration. |
format | Online Article Text |
id | pubmed-8035899 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-80358992021-04-16 Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant Kim, Kibum Kang, Yang Joo Park, Se Ra Kim, Do-Sun Lee, Seung-Won Ko, Kinarm Ponndorf, Daniel Ko, Kisung PeerJ Biophysics Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endoplasmic reticulum retention motif (GA733-FcK) into the Cowpea mosaic virus (CPMV)-based transient plant expression vector, pEAQ-HT. Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ-HT-GA733-Fc and pEAQ-HT-GA733-FcK was infiltrated into the leaves of Nicotiana benthamiana plants. To optimize harvesting of leaf to express therapeutic glycoproteins both spatially and temporally, protein expression levels at various leaf positions (top, middle, and base) and days post-infiltration (dpi) were investigated. The GA733-Fc and GA733-FcK genes were detected in leaves at 1–10 dpi using PCR. As assessed by western blot, GA733-Fc and GA733-FcK were expressed at the highest levels in the top leaf position at 5 dpi, and GA733-FcK was expressed more than GA733-Fc. The proteins were successfully purified from infiltrated N. benthamiana leaves using protein A affinity chromatography. ELISA verified that an anti-GA733 antibody recognized both purified proteins. Thus, a functional GA733-Fc colorectal cancer vaccine protein can be transiently expressed using a CPMV virus-based vector, with an optimized expression time and leaf position post-infiltration. PeerJ Inc. 2021-04-07 /pmc/articles/PMC8035899/ /pubmed/33868796 http://dx.doi.org/10.7717/peerj.10851 Text en © 2021 Kim et al. https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
spellingShingle | Biophysics Kim, Kibum Kang, Yang Joo Park, Se Ra Kim, Do-Sun Lee, Seung-Won Ko, Kinarm Ponndorf, Daniel Ko, Kisung Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant |
title | Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant |
title_full | Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant |
title_fullStr | Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant |
title_full_unstemmed | Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant |
title_short | Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant |
title_sort | effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins ga733-fc and ga733-fck in nicotiana benthamiana plant |
topic | Biophysics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035899/ https://www.ncbi.nlm.nih.gov/pubmed/33868796 http://dx.doi.org/10.7717/peerj.10851 |
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