Heterologous expression, purification and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells
The role of S-layer proteins (SLP), which form the outermost layer of cell walls in lactic acid bacteria (LAB), plays a crucial role in regulating immune-stimulating activity, thereby closely influencing LAB’s ability to boost host immunity. In this study, the heterologous expression, purification,...
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Tsinghua University Press
2025-05-01
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| Series: | Food Science and Human Wellness |
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| Online Access: | https://www.sciopen.com/article/10.26599/FSHW.2024.9250230 |
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| author | Yingying Cao Xiankang Fan Zihang Shi Mingzhen Liu Jue Xu Tao Zhang Xiaoqun Zeng Zhen Wu Daodong Pan |
| author_facet | Yingying Cao Xiankang Fan Zihang Shi Mingzhen Liu Jue Xu Tao Zhang Xiaoqun Zeng Zhen Wu Daodong Pan |
| author_sort | Yingying Cao |
| collection | DOAJ |
| description | The role of S-layer proteins (SLP), which form the outermost layer of cell walls in lactic acid bacteria (LAB), plays a crucial role in regulating immune-stimulating activity, thereby closely influencing LAB’s ability to boost host immunity. In this study, the heterologous expression, purification, and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells were investigated. Initially, the PCR results were shown to successfully clone the slpX DNA sequence by homologous recombination to obtain the pet-32a-slpX recombinant plasmid. SDS-PAGE results revealed that slpX protein with a molecular weight of 54 kDa was successfully obtained under 0.7 mmol/L IPTG-induced conditions, which was further demonstrated by Western blot (WB) and LC-MS/MS. ELISA, WB and molecular docking results indicated that slpX protein can inhibit LPS-induced cellular inflammatory responses by linking to TLR4 and MD2 via hydrogen bonding and increasing the levels of anti-inflammatory factor IL-10 and decreasing the levels of inflammatory factors (TNF-α, IL-6, NO) and ROS via MAPK and NF-κB signaling pathways. The study is essential for the preparation of pure slpX protein and revealing its anti-inflammatory molecular mechanism. |
| format | Article |
| id | doaj-art-ab5fda1a2daa496d9616afc3582f7432 |
| institution | DOAJ |
| issn | 2097-0765 2213-4530 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Tsinghua University Press |
| record_format | Article |
| series | Food Science and Human Wellness |
| spelling | doaj-art-ab5fda1a2daa496d9616afc3582f74322025-08-20T03:12:14ZengTsinghua University PressFood Science and Human Wellness2097-07652213-45302025-05-01145925023010.26599/FSHW.2024.9250230Heterologous expression, purification and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cellsYingying Cao0Xiankang Fan1Zihang Shi2Mingzhen Liu3Jue Xu4Tao Zhang5Xiaoqun Zeng6Zhen Wu7Daodong Pan8State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, ChinaState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, ChinaState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, ChinaState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, ChinaState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, ChinaState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, ChinaState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, ChinaState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, ChinaState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, ChinaThe role of S-layer proteins (SLP), which form the outermost layer of cell walls in lactic acid bacteria (LAB), plays a crucial role in regulating immune-stimulating activity, thereby closely influencing LAB’s ability to boost host immunity. In this study, the heterologous expression, purification, and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells were investigated. Initially, the PCR results were shown to successfully clone the slpX DNA sequence by homologous recombination to obtain the pet-32a-slpX recombinant plasmid. SDS-PAGE results revealed that slpX protein with a molecular weight of 54 kDa was successfully obtained under 0.7 mmol/L IPTG-induced conditions, which was further demonstrated by Western blot (WB) and LC-MS/MS. ELISA, WB and molecular docking results indicated that slpX protein can inhibit LPS-induced cellular inflammatory responses by linking to TLR4 and MD2 via hydrogen bonding and increasing the levels of anti-inflammatory factor IL-10 and decreasing the levels of inflammatory factors (TNF-α, IL-6, NO) and ROS via MAPK and NF-κB signaling pathways. The study is essential for the preparation of pure slpX protein and revealing its anti-inflammatory molecular mechanism.https://www.sciopen.com/article/10.26599/FSHW.2024.9250230lactic acid bacterias-layer proteininflammationliquid chromatography-tandem mass spectrometryenzyme-linked immunosorbnent assaymolecular docking |
| spellingShingle | Yingying Cao Xiankang Fan Zihang Shi Mingzhen Liu Jue Xu Tao Zhang Xiaoqun Zeng Zhen Wu Daodong Pan Heterologous expression, purification and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells Food Science and Human Wellness lactic acid bacteria s-layer protein inflammation liquid chromatography-tandem mass spectrometry enzyme-linked immunosorbnent assay molecular docking |
| title | Heterologous expression, purification and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells |
| title_full | Heterologous expression, purification and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells |
| title_fullStr | Heterologous expression, purification and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells |
| title_full_unstemmed | Heterologous expression, purification and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells |
| title_short | Heterologous expression, purification and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells |
| title_sort | heterologous expression purification and characterization of lactobacillus acidophilus cicc6074 derived slpx protein and the molecular mechanism of its anti inflammatory effect on lps induced raw264 7 cells |
| topic | lactic acid bacteria s-layer protein inflammation liquid chromatography-tandem mass spectrometry enzyme-linked immunosorbnent assay molecular docking |
| url | https://www.sciopen.com/article/10.26599/FSHW.2024.9250230 |
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