<i>Escherichia coli</i>-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles
Tandem-core hepatitis B core antigen (HBcAg) virus-like particles (VLPs), in which two HBcAg monomers are joined together by a peptide linker, can be used to display two different antigens on the VLP surface. We produced universal influenza vaccine candidates that use this scaffold in an <i>Es...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-02-01
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| Series: | Vaccines |
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| Online Access: | https://www.mdpi.com/2076-393X/9/3/193 |
| _version_ | 1797395227039760384 |
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| author | Noelle Colant Beatrice Melinek Stefanie Frank William Rosenberg Daniel G. Bracewell |
| author_facet | Noelle Colant Beatrice Melinek Stefanie Frank William Rosenberg Daniel G. Bracewell |
| author_sort | Noelle Colant |
| collection | DOAJ |
| description | Tandem-core hepatitis B core antigen (HBcAg) virus-like particles (VLPs), in which two HBcAg monomers are joined together by a peptide linker, can be used to display two different antigens on the VLP surface. We produced universal influenza vaccine candidates that use this scaffold in an <i>Escherichia coli</i>-based cell-free protein synthesis (CFPS) platform. We then used the CFPS system to rapidly test modifications to the arginine-rich region typically found in wild-type HBcAg, the peptide linkers around the influenza antigen inserts, and the plasmid vector backbone to improve titer and quality. Using a minimal plasmid vector backbone designed for CFPS improved titers by at least 1.4-fold over the original constructs. When the linker lengths for the influenza inserts were more consistent in length and a greater variety of codons for glycine and serine were utilized, titers were further increased to over 70 μg/mL (4.0-fold greater than the original construct) and the presence of lower molecular weight product-related impurities was significantly reduced, although improvements in particle assembly were not seen. Furthermore, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. This demonstrates the potential for CFPS as a screening platform for VLPs. |
| first_indexed | 2024-03-09T00:31:23Z |
| format | Article |
| id | doaj.art-54298a21d3004d84a08dc9d3e5c1d6ad |
| institution | Directory Open Access Journal |
| issn | 2076-393X |
| language | English |
| last_indexed | 2024-03-09T00:31:23Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Vaccines |
| spelling | doaj.art-54298a21d3004d84a08dc9d3e5c1d6ad2023-12-11T18:29:52ZengMDPI AGVaccines2076-393X2021-02-019319310.3390/vaccines9030193<i>Escherichia coli</i>-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like ParticlesNoelle Colant0Beatrice Melinek1Stefanie Frank2William Rosenberg3Daniel G. Bracewell4Department of Biochemical Engineering, University College London, London WC1E 6BT, UKDepartment of Biochemical Engineering, University College London, London WC1E 6BT, UKDepartment of Biochemical Engineering, University College London, London WC1E 6BT, UKDivision of Medicine, UCL Institute for Liver and Digestive Health, Royal Free Campus, London NW3 2PF, UKDepartment of Biochemical Engineering, University College London, London WC1E 6BT, UKTandem-core hepatitis B core antigen (HBcAg) virus-like particles (VLPs), in which two HBcAg monomers are joined together by a peptide linker, can be used to display two different antigens on the VLP surface. We produced universal influenza vaccine candidates that use this scaffold in an <i>Escherichia coli</i>-based cell-free protein synthesis (CFPS) platform. We then used the CFPS system to rapidly test modifications to the arginine-rich region typically found in wild-type HBcAg, the peptide linkers around the influenza antigen inserts, and the plasmid vector backbone to improve titer and quality. Using a minimal plasmid vector backbone designed for CFPS improved titers by at least 1.4-fold over the original constructs. When the linker lengths for the influenza inserts were more consistent in length and a greater variety of codons for glycine and serine were utilized, titers were further increased to over 70 μg/mL (4.0-fold greater than the original construct) and the presence of lower molecular weight product-related impurities was significantly reduced, although improvements in particle assembly were not seen. Furthermore, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. This demonstrates the potential for CFPS as a screening platform for VLPs.https://www.mdpi.com/2076-393X/9/3/193cell-free protein synthesisvirus-like particletandem-coreinfluenza vaccine |
| spellingShingle | Noelle Colant Beatrice Melinek Stefanie Frank William Rosenberg Daniel G. Bracewell <i>Escherichia coli</i>-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles Vaccines cell-free protein synthesis virus-like particle tandem-core influenza vaccine |
| title | <i>Escherichia coli</i>-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles |
| title_full | <i>Escherichia coli</i>-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles |
| title_fullStr | <i>Escherichia coli</i>-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles |
| title_full_unstemmed | <i>Escherichia coli</i>-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles |
| title_short | <i>Escherichia coli</i>-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles |
| title_sort | i escherichia coli i based cell free protein synthesis for iterative design of tandem core virus like particles |
| topic | cell-free protein synthesis virus-like particle tandem-core influenza vaccine |
| url | https://www.mdpi.com/2076-393X/9/3/193 |
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