Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence—An Experimental Study of the Human Voltage-Dependent Anion Channel
Membrane proteins are involved in many aspects of cellular biology; for example, they regulate how cells interact with their environment, so such proteins are important drug targets. The rapid advancement in the field of immune effector cell therapy has been expanding the horizons of synthetic membr...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-09-01
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| Series: | Membranes |
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| Online Access: | https://www.mdpi.com/2077-0375/11/10/741 |
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| author | Sonja Zayni Samar Damiati Susana Moreno-Flores Fabian Amman Ivo Hofacker David Jin Eva-Kathrin Ehmoser |
| author_facet | Sonja Zayni Samar Damiati Susana Moreno-Flores Fabian Amman Ivo Hofacker David Jin Eva-Kathrin Ehmoser |
| author_sort | Sonja Zayni |
| collection | DOAJ |
| description | Membrane proteins are involved in many aspects of cellular biology; for example, they regulate how cells interact with their environment, so such proteins are important drug targets. The rapid advancement in the field of immune effector cell therapy has been expanding the horizons of synthetic membrane receptors in the areas of cell-based immunotherapy and cellular medicine. However, the investigation of membrane proteins, which are key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, which is of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage-dependent anion channel (VDAC), which is a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function that quantifies the feasibility of the formation of the translation initiation complex through the ribosome–mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome is proposed. The scoring function enables one to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies. This scoring function is publicly available as webservice XenoExpressO at University of Vienna, Austria. |
| first_indexed | 2024-03-10T06:23:56Z |
| format | Article |
| id | doaj.art-5cd2d62910aa41d98c4e3a0790948c45 |
| institution | Directory Open Access Journal |
| issn | 2077-0375 |
| language | English |
| last_indexed | 2024-03-10T06:23:56Z |
| publishDate | 2021-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Membranes |
| spelling | doaj.art-5cd2d62910aa41d98c4e3a0790948c452023-11-22T19:05:55ZengMDPI AGMembranes2077-03752021-09-01111074110.3390/membranes11100741Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence—An Experimental Study of the Human Voltage-Dependent Anion ChannelSonja Zayni0Samar Damiati1Susana Moreno-Flores2Fabian Amman3Ivo Hofacker4David Jin5Eva-Kathrin Ehmoser6Department of Nanobiotechnology, Institute for Synthetic Bioarchitectures, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 11, A-1190 Wien, AustriaDepartment of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21413, Saudi ArabiaIndependent Researcher, A-1190 Vienna, AustriaDepartment of Theoretical Chemistry, University of Vienna, Währinger Straße 17, A-1090 Wien, AustriaDepartment of Theoretical Chemistry, University of Vienna, Währinger Straße 17, A-1090 Wien, AustriaAvalon Globocare Corp., 4400 Route 9 South, Suite 3100, Freehold, NJ 07728, USADepartment of Nanobiotechnology, Institute for Synthetic Bioarchitectures, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 11, A-1190 Wien, AustriaMembrane proteins are involved in many aspects of cellular biology; for example, they regulate how cells interact with their environment, so such proteins are important drug targets. The rapid advancement in the field of immune effector cell therapy has been expanding the horizons of synthetic membrane receptors in the areas of cell-based immunotherapy and cellular medicine. However, the investigation of membrane proteins, which are key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, which is of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage-dependent anion channel (VDAC), which is a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function that quantifies the feasibility of the formation of the translation initiation complex through the ribosome–mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome is proposed. The scoring function enables one to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies. This scoring function is publicly available as webservice XenoExpressO at University of Vienna, Austria.https://www.mdpi.com/2077-0375/11/10/741cell-free membrane protein expressiontranslation enhancertranslation initiationribosome docking sitesequence design |
| spellingShingle | Sonja Zayni Samar Damiati Susana Moreno-Flores Fabian Amman Ivo Hofacker David Jin Eva-Kathrin Ehmoser Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence—An Experimental Study of the Human Voltage-Dependent Anion Channel Membranes cell-free membrane protein expression translation enhancer translation initiation ribosome docking site sequence design |
| title | Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence—An Experimental Study of the Human Voltage-Dependent Anion Channel |
| title_full | Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence—An Experimental Study of the Human Voltage-Dependent Anion Channel |
| title_fullStr | Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence—An Experimental Study of the Human Voltage-Dependent Anion Channel |
| title_full_unstemmed | Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence—An Experimental Study of the Human Voltage-Dependent Anion Channel |
| title_short | Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence—An Experimental Study of the Human Voltage-Dependent Anion Channel |
| title_sort | enhancing the cell free expression of native membrane proteins by in silico optimization of the coding sequence an experimental study of the human voltage dependent anion channel |
| topic | cell-free membrane protein expression translation enhancer translation initiation ribosome docking site sequence design |
| url | https://www.mdpi.com/2077-0375/11/10/741 |
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