Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approach
HEK293T cells are extensively utilized for therapeutic protein production due to their human origin, which enables accurate post-translational modifications. This study aimed to enhance membrane protein production in HEK293T cells by knocking out the ATF4 gene using CRISPR-Cas9 technology. The ATF4...
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Association of Basic Medical Sciences of Federation of Bosnia and Herzegovina
2025-01-01
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| Series: | Biomolecules & Biomedicine |
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| Online Access: | https://www.bjbms.org/ojs/index.php/bjbms/article/view/11519 |
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| author | Byung-Jo Choi Ba Reum Kim Ho Joong Choi Ok-Hee Kim Say-June Kim |
| author_facet | Byung-Jo Choi Ba Reum Kim Ho Joong Choi Ok-Hee Kim Say-June Kim |
| author_sort | Byung-Jo Choi |
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HEK293T cells are extensively utilized for therapeutic protein production due to their human origin, which enables accurate post-translational modifications. This study aimed to enhance membrane protein production in HEK293T cells by knocking out the ATF4 gene using CRISPR-Cas9 technology. The ATF4 gene was edited by infecting HEK293T cells with a lentivirus carrying optimized single-guide RNA (ATF4-KO-3) and Cas9 genes. Comparative evaluations were conducted using all-in-one and two-vector systems. Genome sequencing and membrane protein productivity of ATF4-KO cells were compared to wild-type cells using next-generation sequencing (NGS) and a membrane protein isolation kit, respectively. Single-cell analysis confirmed gene editing patterns, with NGS verifying the intended deletions. Membrane protein production was also assessed indirectly via flow cytometry, analyzing cells expressing Membrane-GFP. Compared to wild-type cells, ATF4-KO cells exhibited a significant increase in membrane protein production, with a 52.2 ± 19.0% improvement. Gene editing efficiency was compared between the two delivery systems, with the two-vector system demonstrating higher efficiency based on T7E1 assays. Western blot analysis confirmed ATF4 suppression and increased expression of membrane proteins, including E-cadherin and CD63. Quantitative analysis via PAGE revealed a 77.2 ± 30.6% increase in purified membrane protein yields, consistent with the observed enhancements. Flow cytometry using Membrane-GFP further demonstrated a 22.9 ± 9.7% increase in productivity. In summary, ATF4 knockout significantly enhances membrane protein production in HEK293T cells, offering potential improvements in biopharmaceutical manufacturing by enabling more efficient protein synthesis.
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| format | Article |
| id | doaj-art-77f429b36aee4d10a521111a6ddf3628 |
| institution | Kabale University |
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| language | English |
| publishDate | 2025-01-01 |
| publisher | Association of Basic Medical Sciences of Federation of Bosnia and Herzegovina |
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| series | Biomolecules & Biomedicine |
| spelling | doaj-art-77f429b36aee4d10a521111a6ddf36282025-01-22T16:40:58ZengAssociation of Basic Medical Sciences of Federation of Bosnia and HerzegovinaBiomolecules & Biomedicine2831-08962831-090X2025-01-0110.17305/bb.2024.11519Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approachByung-Jo Choi0Ba Reum Kim1Ho Joong Choi2Ok-Hee Kim3Say-June Kim4Department of Surgery, Daejeon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Catholic Central Laboratory of Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of KoreaTranslational Research Team, Surginex Co., Republic of KoreaDepartment of Surgery, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of KoreaCatholic Central Laboratory of Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Translational Research Team, Surginex Co., Republic of KoreaCatholic Central Laboratory of Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Translational Research Team, Surginex Co., Republic of Korea; Department of Surgery, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea HEK293T cells are extensively utilized for therapeutic protein production due to their human origin, which enables accurate post-translational modifications. This study aimed to enhance membrane protein production in HEK293T cells by knocking out the ATF4 gene using CRISPR-Cas9 technology. The ATF4 gene was edited by infecting HEK293T cells with a lentivirus carrying optimized single-guide RNA (ATF4-KO-3) and Cas9 genes. Comparative evaluations were conducted using all-in-one and two-vector systems. Genome sequencing and membrane protein productivity of ATF4-KO cells were compared to wild-type cells using next-generation sequencing (NGS) and a membrane protein isolation kit, respectively. Single-cell analysis confirmed gene editing patterns, with NGS verifying the intended deletions. Membrane protein production was also assessed indirectly via flow cytometry, analyzing cells expressing Membrane-GFP. Compared to wild-type cells, ATF4-KO cells exhibited a significant increase in membrane protein production, with a 52.2 ± 19.0% improvement. Gene editing efficiency was compared between the two delivery systems, with the two-vector system demonstrating higher efficiency based on T7E1 assays. Western blot analysis confirmed ATF4 suppression and increased expression of membrane proteins, including E-cadherin and CD63. Quantitative analysis via PAGE revealed a 77.2 ± 30.6% increase in purified membrane protein yields, consistent with the observed enhancements. Flow cytometry using Membrane-GFP further demonstrated a 22.9 ± 9.7% increase in productivity. In summary, ATF4 knockout significantly enhances membrane protein production in HEK293T cells, offering potential improvements in biopharmaceutical manufacturing by enabling more efficient protein synthesis. https://www.bjbms.org/ojs/index.php/bjbms/article/view/11519CRISPR-Cas9gene editingHEK293T cellsATF4 knockoutmembrane protein production |
| spellingShingle | Byung-Jo Choi Ba Reum Kim Ho Joong Choi Ok-Hee Kim Say-June Kim Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approach Biomolecules & Biomedicine CRISPR-Cas9 gene editing HEK293T cells ATF4 knockout membrane protein production |
| title | Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approach |
| title_full | Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approach |
| title_fullStr | Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approach |
| title_full_unstemmed | Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approach |
| title_short | Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approach |
| title_sort | enhanced membrane protein production in hek293t cells via atf4 gene knockout a crispr cas9 mediated approach |
| topic | CRISPR-Cas9 gene editing HEK293T cells ATF4 knockout membrane protein production |
| url | https://www.bjbms.org/ojs/index.php/bjbms/article/view/11519 |
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