Per citar aquest document: http://ddd.uab.cat/record/112890
Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris
Surribas i Casalprim, Anna (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
Resina Rodríguez, David (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
Ferrer i Alegre, Pau (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
Valero Barranco, Francisco (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)

Data: 2007
Resum: Background: 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 Rhizopus oryzae lipase (ROL) in Pichia pastoris by means of 2D-fluorimetric techniques. Results: In this study, the GFP-ROL fusion protein was successfully produced as a secreted fusion form in P. pastoris 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. Conclusion: P. pastoris appears to secrete/excrete significant amounts of riboflavin to the culture medium. When attempting to monitor extracellular protein production in P. pastoris 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.
Drets: Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial i la comunicació pública de l'obra, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original. No es permet la creació d'obres derivades. Creative Commons
Llengua: Anglès.
Document: article ; publishedVersion
Publicat a: Microbial cell factories, Vol. 6, N. 15 (May 2007) , p. 1-7, ISSN 1475-2859

DOI: 10.1186/1475-2859-6-15


7 p, 441.8 KB

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