Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in <it>Pichia pastoris</it>
<p>Abstract</p> <p>Background</p> <p>Together with the development of optical sensors, fluorometry is becoming an increasingly attractive tool for the monitoring of cultivation processes. In this context, the green fluorescence protein (GFP) has been proposed as a molec...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2007-05-01
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| Series: | Microbial Cell Factories |
| Online Access: | http://www.microbialcellfactories.com/content/6/1/15 |
| _version_ | 1811297032784052224 |
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| author | Valero Francisco Ferrer Pau Resina David Surribas Anna |
| author_facet | Valero Francisco Ferrer Pau Resina David Surribas Anna |
| author_sort | Valero Francisco |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Together with the development of optical sensors, fluorometry is becoming an increasingly attractive tool for the monitoring of cultivation processes. In this context, the green fluorescence protein (GFP) has been proposed as a molecular reporter when fused to target proteins to study their subcellular localization or secretion behaviour. The present work evaluates the use of the GFP fusion partner for monitoring extracellular production of a <it>Rhizopus oryzae </it>lipase (ROL) in <it>Pichia pastoris </it>by means of 2D-fluorimetric techniques</p> <p>Results</p> <p>In this study, the GFP-ROL fusion protein was successfully produced as a secreted fusion form in <it>P. pastoris </it>batch cultivations. Furthermore, both the fusion enzyme and the fluorescent protein (GFP S65T mutant) retained their biological activity. However, when multiwavelength spectrofluorometry was used for extracellular fusion protein monitoring, riboflavin appeared as a major interfering component with GFP signal. Only when riboflavin was removed by ultrafiltration from cultivation supernatants, GFP fluorescence signal linearly correlated to lipase activity</p> <p>Conclusion</p> <p><it>P. pastoris </it>appears to secrete/excrete significant amounts of riboflavin to the culture medium. When attempting to monitor extracellular protein production in <it>P. pastoris </it>using GFP fusions combined with multiwavelength spectrofluorimetric techniques, riboflavin may interfere with GFP fluorescence signal, thus limiting the application of some GFP variants for on-line extracellular recombinant protein quantification and monitoring purposes.</p> |
| first_indexed | 2024-04-13T05:58:05Z |
| format | Article |
| id | doaj.art-bdf6fe8d16a64748ba15b3686fd3f7e6 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-04-13T05:58:05Z |
| publishDate | 2007-05-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-bdf6fe8d16a64748ba15b3686fd3f7e62022-12-22T02:59:34ZengBMCMicrobial Cell Factories1475-28592007-05-01611510.1186/1475-2859-6-15Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in <it>Pichia pastoris</it>Valero FranciscoFerrer PauResina DavidSurribas Anna<p>Abstract</p> <p>Background</p> <p>Together with the development of optical sensors, fluorometry is becoming an increasingly attractive tool for the monitoring of cultivation processes. In this context, the green fluorescence protein (GFP) has been proposed as a molecular reporter when fused to target proteins to study their subcellular localization or secretion behaviour. The present work evaluates the use of the GFP fusion partner for monitoring extracellular production of a <it>Rhizopus oryzae </it>lipase (ROL) in <it>Pichia pastoris </it>by means of 2D-fluorimetric techniques</p> <p>Results</p> <p>In this study, the GFP-ROL fusion protein was successfully produced as a secreted fusion form in <it>P. pastoris </it>batch cultivations. Furthermore, both the fusion enzyme and the fluorescent protein (GFP S65T mutant) retained their biological activity. However, when multiwavelength spectrofluorometry was used for extracellular fusion protein monitoring, riboflavin appeared as a major interfering component with GFP signal. Only when riboflavin was removed by ultrafiltration from cultivation supernatants, GFP fluorescence signal linearly correlated to lipase activity</p> <p>Conclusion</p> <p><it>P. pastoris </it>appears to secrete/excrete significant amounts of riboflavin to the culture medium. When attempting to monitor extracellular protein production in <it>P. pastoris </it>using GFP fusions combined with multiwavelength spectrofluorimetric techniques, riboflavin may interfere with GFP fluorescence signal, thus limiting the application of some GFP variants for on-line extracellular recombinant protein quantification and monitoring purposes.</p>http://www.microbialcellfactories.com/content/6/1/15 |
| spellingShingle | Valero Francisco Ferrer Pau Resina David Surribas Anna Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in <it>Pichia pastoris</it> Microbial Cell Factories |
| title | Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in <it>Pichia pastoris</it> |
| title_full | Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in <it>Pichia pastoris</it> |
| title_fullStr | Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in <it>Pichia pastoris</it> |
| title_full_unstemmed | Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in <it>Pichia pastoris</it> |
| title_short | Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in <it>Pichia pastoris</it> |
| title_sort | rivoflavin may interfere with on line monitoring of secreted green fluorescence protein fusion proteins in it pichia pastoris it |
| url | http://www.microbialcellfactories.com/content/6/1/15 |
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