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A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts
We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassett...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936851/ https://www.ncbi.nlm.nih.gov/pubmed/31887188 http://dx.doi.org/10.1371/journal.pone.0227110 |
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author | Somogyi, Márk Szimler, Tamás Baksa, Attila Végh, Barbara M. Bakos, Tamás Paréj, Katalin Ádám, Csaba Zsigmond, Áron Megyeri, Márton Flachner, Beáta Sajó, Ráchel Gráczer, Éva Závodszky, Péter Hajdú, István Beinrohr, László |
author_facet | Somogyi, Márk Szimler, Tamás Baksa, Attila Végh, Barbara M. Bakos, Tamás Paréj, Katalin Ádám, Csaba Zsigmond, Áron Megyeri, Márton Flachner, Beáta Sajó, Ráchel Gráczer, Éva Závodszky, Péter Hajdú, István Beinrohr, László |
author_sort | Somogyi, Márk |
collection | PubMed |
description | We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set. |
format | Online Article Text |
id | pubmed-6936851 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-69368512020-01-07 A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts Somogyi, Márk Szimler, Tamás Baksa, Attila Végh, Barbara M. Bakos, Tamás Paréj, Katalin Ádám, Csaba Zsigmond, Áron Megyeri, Márton Flachner, Beáta Sajó, Ráchel Gráczer, Éva Závodszky, Péter Hajdú, István Beinrohr, László PLoS One Research Article We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set. Public Library of Science 2019-12-30 /pmc/articles/PMC6936851/ /pubmed/31887188 http://dx.doi.org/10.1371/journal.pone.0227110 Text en © 2019 Somogyi et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Somogyi, Márk Szimler, Tamás Baksa, Attila Végh, Barbara M. Bakos, Tamás Paréj, Katalin Ádám, Csaba Zsigmond, Áron Megyeri, Márton Flachner, Beáta Sajó, Ráchel Gráczer, Éva Závodszky, Péter Hajdú, István Beinrohr, László A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts |
title | A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts |
title_full | A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts |
title_fullStr | A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts |
title_full_unstemmed | A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts |
title_short | A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts |
title_sort | versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936851/ https://www.ncbi.nlm.nih.gov/pubmed/31887188 http://dx.doi.org/10.1371/journal.pone.0227110 |
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