Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles
All Gram-negative bacteria are believed to produce outer membrane vesicles (OMVs), proteoliposomes shed from the outermost membrane. We previously separately engineered <i>E. coli</i> to produce and package two organophosphate (OP) hydrolyzing enzymes, phosphotriesterase (PTE) and diisop...
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MDPI AG
2023-05-01
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| Series: | Bioengineering |
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| Online Access: | https://www.mdpi.com/2306-5354/10/5/583 |
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| author | Scott N. Dean Meghna Thakur Joseph R. Spangler Aaron D. Smith Sean P. Garin Scott A. Walper Gregory A. Ellis |
| author_facet | Scott N. Dean Meghna Thakur Joseph R. Spangler Aaron D. Smith Sean P. Garin Scott A. Walper Gregory A. Ellis |
| author_sort | Scott N. Dean |
| collection | DOAJ |
| description | All Gram-negative bacteria are believed to produce outer membrane vesicles (OMVs), proteoliposomes shed from the outermost membrane. We previously separately engineered <i>E. coli</i> to produce and package two organophosphate (OP) hydrolyzing enzymes, phosphotriesterase (PTE) and diisopropylfluorophosphatase (DFPase), into secreted OMVs. From this work, we realized a need to thoroughly compare multiple packaging strategies to elicit design rules for this process, focused on (1) membrane anchors or periplasm-directing proteins (herein “anchors/directors”) and (2) the linkers connecting these to the cargo enzyme; both may affect enzyme cargo activity. Herein, we assessed six anchors/directors to load PTE and DFPase into OMVs: four membrane anchors, namely, lipopeptide Lpp’, SlyB, SLP, and OmpA, and two periplasm-directing proteins, namely, maltose-binding protein (MBP) and BtuF. To test the effect of linker length and rigidity, four different linkers were compared using the anchor Lpp’. Our results showed that PTE and DFPase were packaged with most anchors/directors to different degrees. For the Lpp’ anchor, increased packaging and activity corresponded to increased linker length. Our findings demonstrate that the selection of anchors/directors and linkers can greatly influence the packaging and bioactivity of enzymes loaded into OMVs, and these findings have the potential to be utilized for packaging other enzymes into OMVs. |
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| last_indexed | 2024-03-11T03:55:23Z |
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| series | Bioengineering |
| spelling | doaj.art-126f58f649f74a0c85b1bfcf2c55a68d2023-11-18T00:31:37ZengMDPI AGBioengineering2306-53542023-05-0110558310.3390/bioengineering10050583Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane VesiclesScott N. Dean0Meghna Thakur1Joseph R. Spangler2Aaron D. Smith3Sean P. Garin4Scott A. Walper5Gregory A. Ellis6Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, USACollege of Science, George Mason University, Fairfax, VA 22030, USACenter for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, USACenter for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, USACenter for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, USACenter for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, USACenter for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, USAAll Gram-negative bacteria are believed to produce outer membrane vesicles (OMVs), proteoliposomes shed from the outermost membrane. We previously separately engineered <i>E. coli</i> to produce and package two organophosphate (OP) hydrolyzing enzymes, phosphotriesterase (PTE) and diisopropylfluorophosphatase (DFPase), into secreted OMVs. From this work, we realized a need to thoroughly compare multiple packaging strategies to elicit design rules for this process, focused on (1) membrane anchors or periplasm-directing proteins (herein “anchors/directors”) and (2) the linkers connecting these to the cargo enzyme; both may affect enzyme cargo activity. Herein, we assessed six anchors/directors to load PTE and DFPase into OMVs: four membrane anchors, namely, lipopeptide Lpp’, SlyB, SLP, and OmpA, and two periplasm-directing proteins, namely, maltose-binding protein (MBP) and BtuF. To test the effect of linker length and rigidity, four different linkers were compared using the anchor Lpp’. Our results showed that PTE and DFPase were packaged with most anchors/directors to different degrees. For the Lpp’ anchor, increased packaging and activity corresponded to increased linker length. Our findings demonstrate that the selection of anchors/directors and linkers can greatly influence the packaging and bioactivity of enzymes loaded into OMVs, and these findings have the potential to be utilized for packaging other enzymes into OMVs.https://www.mdpi.com/2306-5354/10/5/583outer membrane vesicles (OMVs)phosphotriesterase (PTE)diisopropyl fluorophosphatase (DFPase) |
| spellingShingle | Scott N. Dean Meghna Thakur Joseph R. Spangler Aaron D. Smith Sean P. Garin Scott A. Walper Gregory A. Ellis Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles Bioengineering outer membrane vesicles (OMVs) phosphotriesterase (PTE) diisopropyl fluorophosphatase (DFPase) |
| title | Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles |
| title_full | Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles |
| title_fullStr | Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles |
| title_full_unstemmed | Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles |
| title_short | Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles |
| title_sort | different strategies affect enzyme packaging into bacterial outer membrane vesicles |
| topic | outer membrane vesicles (OMVs) phosphotriesterase (PTE) diisopropyl fluorophosphatase (DFPase) |
| url | https://www.mdpi.com/2306-5354/10/5/583 |
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