Modified recombinant proteins can be exported via the Sec pathway in Escherichia coli.
The correct folding of a protein is a pre-requirement for its proper posttranslational modification. The Escherichia coli Sec pathway, in which preproteins, in an unfolded, translocation-competent state, are rapidly secreted across the cytoplasmic membrane, is commonly assumed to be unfavorable for...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3418276?pdf=render |
| _version_ | 1811324983662608384 |
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| author | Nan Chen Fu-Lin Hong Hai-Hong Wang Qi-Hang Yuan Wan-Yan Ma Xu-Na Gao Rui Shi Rui-Juan Zhang Chang-Sheng Sun Sheng-Bin Wang |
| author_facet | Nan Chen Fu-Lin Hong Hai-Hong Wang Qi-Hang Yuan Wan-Yan Ma Xu-Na Gao Rui Shi Rui-Juan Zhang Chang-Sheng Sun Sheng-Bin Wang |
| author_sort | Nan Chen |
| collection | DOAJ |
| description | The correct folding of a protein is a pre-requirement for its proper posttranslational modification. The Escherichia coli Sec pathway, in which preproteins, in an unfolded, translocation-competent state, are rapidly secreted across the cytoplasmic membrane, is commonly assumed to be unfavorable for their modification in the cytosol. Whether posttranslationally modified recombinant preproteins can be efficiently transported via the Sec pathway, however, remains unclear. ACP and BCCP domain (BCCP87) are carrier proteins that can be converted into active phosphopantetheinylated ACP (holo-ACP) and biotinylated-BCCP (holo-BCCP) by AcpS and BirA, respectively. In the present study, we show that, when ACP or BCCP87 is fused to the C-terminus of secretory protein YebF or MBP, the resulting fusion protein preYebF-ACP, preYebF-BCCP87, preMBP-ACP or preMBP-BCCP87 can be modified and then secreted. Our data demonstrate that posttranslational modification of preYebF-ACP, preYebF-BCCP87 preMBP-ACP and preMBP-BCCP87 can take place in the cytosol prior to translocation, and the Sec machinery accommodates these previously modified fusion proteins. High levels of active holo-ACP and holo-BCCP87 are achieved when AcpS or BirA is co-expressed, especially when sodium azide is used to retard their translocation across the inner membrane. Our results also provide an alternative to achieve a high level of modified recombinant proteins expressed extracellularly. |
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| format | Article |
| id | doaj.art-678f00e6205c4640ab851f0b8c34a0c8 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T14:25:12Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-678f00e6205c4640ab851f0b8c34a0c82022-12-22T02:43:20ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0178e4251910.1371/journal.pone.0042519Modified recombinant proteins can be exported via the Sec pathway in Escherichia coli.Nan ChenFu-Lin HongHai-Hong WangQi-Hang YuanWan-Yan MaXu-Na GaoRui ShiRui-Juan ZhangChang-Sheng SunSheng-Bin WangThe correct folding of a protein is a pre-requirement for its proper posttranslational modification. The Escherichia coli Sec pathway, in which preproteins, in an unfolded, translocation-competent state, are rapidly secreted across the cytoplasmic membrane, is commonly assumed to be unfavorable for their modification in the cytosol. Whether posttranslationally modified recombinant preproteins can be efficiently transported via the Sec pathway, however, remains unclear. ACP and BCCP domain (BCCP87) are carrier proteins that can be converted into active phosphopantetheinylated ACP (holo-ACP) and biotinylated-BCCP (holo-BCCP) by AcpS and BirA, respectively. In the present study, we show that, when ACP or BCCP87 is fused to the C-terminus of secretory protein YebF or MBP, the resulting fusion protein preYebF-ACP, preYebF-BCCP87, preMBP-ACP or preMBP-BCCP87 can be modified and then secreted. Our data demonstrate that posttranslational modification of preYebF-ACP, preYebF-BCCP87 preMBP-ACP and preMBP-BCCP87 can take place in the cytosol prior to translocation, and the Sec machinery accommodates these previously modified fusion proteins. High levels of active holo-ACP and holo-BCCP87 are achieved when AcpS or BirA is co-expressed, especially when sodium azide is used to retard their translocation across the inner membrane. Our results also provide an alternative to achieve a high level of modified recombinant proteins expressed extracellularly.http://europepmc.org/articles/PMC3418276?pdf=render |
| spellingShingle | Nan Chen Fu-Lin Hong Hai-Hong Wang Qi-Hang Yuan Wan-Yan Ma Xu-Na Gao Rui Shi Rui-Juan Zhang Chang-Sheng Sun Sheng-Bin Wang Modified recombinant proteins can be exported via the Sec pathway in Escherichia coli. PLoS ONE |
| title | Modified recombinant proteins can be exported via the Sec pathway in Escherichia coli. |
| title_full | Modified recombinant proteins can be exported via the Sec pathway in Escherichia coli. |
| title_fullStr | Modified recombinant proteins can be exported via the Sec pathway in Escherichia coli. |
| title_full_unstemmed | Modified recombinant proteins can be exported via the Sec pathway in Escherichia coli. |
| title_short | Modified recombinant proteins can be exported via the Sec pathway in Escherichia coli. |
| title_sort | modified recombinant proteins can be exported via the sec pathway in escherichia coli |
| url | http://europepmc.org/articles/PMC3418276?pdf=render |
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