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Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris.

Surribas A, Resina D, Ferrer P, Valero F - Microb. Cell Fact. (2007)

Bottom Line: 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.Only when riboflavin was removed by ultrafiltration from cultivation supernatants, GFP fluorescence signal linearly correlated to lipase activity P. pastoris appears to secrete/excrete significant amounts of riboflavin to the culture medium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departament d'Enginyeria Química, Escola Tècnica Superior d'Enginyeria, Universitat Autònoma de Barcelona, 08193-Bellaterra (Cerdanyola del Vallès), Spain. anna.surribas@uab.cat

ABSTRACT

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.

No MeSH data available.


Lipase activity versus GFP fluorescence correlations in ultrafiltrated culture supernatants. Correlation between the measured lipolytic activity and the relative fluorescence in ultrafiltrated culture samples (●). Excitation and emission wavelengths were 489 nm and 510 nm respectively.
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Figure 4: Lipase activity versus GFP fluorescence correlations in ultrafiltrated culture supernatants. Correlation between the measured lipolytic activity and the relative fluorescence in ultrafiltrated culture samples (●). Excitation and emission wavelengths were 489 nm and 510 nm respectively.

Mentions: Importantly, extracellular GFP fluorescence and lipase activity from ultrafiltred samples were linearly correlated (figure 4), i.e. showing the potential of GFP fusions for on-line quantitative monitoring of ROL secretion in P. pastoris using fluorometric techniques, even in the case where secreted product titers may be low.


Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris.

Surribas A, Resina D, Ferrer P, Valero F - Microb. Cell Fact. (2007)

Lipase activity versus GFP fluorescence correlations in ultrafiltrated culture supernatants. Correlation between the measured lipolytic activity and the relative fluorescence in ultrafiltrated culture samples (●). Excitation and emission wavelengths were 489 nm and 510 nm respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1884171&req=5

Figure 4: Lipase activity versus GFP fluorescence correlations in ultrafiltrated culture supernatants. Correlation between the measured lipolytic activity and the relative fluorescence in ultrafiltrated culture samples (●). Excitation and emission wavelengths were 489 nm and 510 nm respectively.
Mentions: Importantly, extracellular GFP fluorescence and lipase activity from ultrafiltred samples were linearly correlated (figure 4), i.e. showing the potential of GFP fusions for on-line quantitative monitoring of ROL secretion in P. pastoris using fluorometric techniques, even in the case where secreted product titers may be low.

Bottom Line: 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.Only when riboflavin was removed by ultrafiltration from cultivation supernatants, GFP fluorescence signal linearly correlated to lipase activity P. pastoris appears to secrete/excrete significant amounts of riboflavin to the culture medium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departament d'Enginyeria Química, Escola Tècnica Superior d'Enginyeria, Universitat Autònoma de Barcelona, 08193-Bellaterra (Cerdanyola del Vallès), Spain. anna.surribas@uab.cat

ABSTRACT

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.

No MeSH data available.