Potential of real-time measurement of GFP-fusion proteins

Building on the basic design concepts of Randers-Eichhorn [Biotechnol. Bioeng. 55 (1997) 921], an on-line, real-time robust, steam sterilisable optical sensor for monitoring green fluorescent protein (GFP) has been developed. A general cloning vector for fusion expression proteins was constructed, a...

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Bibliographic Details
Published in:	Journal of biotechnology Vol. 109; no. 1; pp. 201 - 211
Main Authors:	Jones, Jo J, Bridges, Angela M, Fosberry, Andrew P, Gardner, Sharmila, Lowers, Robert R, Newby, Rachel R, James, Philip J, Hall, Richard M, Jenkins, Owen
Format:	Journal Article Conference Proceeding
Language:	English
Published:	Lausanne Elsevier B.V 08-04-2004 Amsterdam Elsevier New York, NY
Subjects:	Biological and medical sciences Biosensing Techniques - instrumentation Biotechnology Fermentation Fundamental and applied biological sciences. Psychology Genetic Vectors - genetics Green fluorescent protein (GFP) On-line sensor Recombinant Fusion Proteins - analysis Recombinant Fusion Proteins - biosynthesis Recombinant protein expression On-line sensor Green fluorescent protein (GFP) Recombinant protein expression Fusion protein Real time Green fluorescent protein
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Summary:	Building on the basic design concepts of Randers-Eichhorn [Biotechnol. Bioeng. 55 (1997) 921], an on-line, real-time robust, steam sterilisable optical sensor for monitoring green fluorescent protein (GFP) has been developed. A general cloning vector for fusion expression proteins was constructed, allowing expression of both GFP and the target protein as a fusion. Cultivations were carried out at the 20 l scale with the signal from the sensor being relayed directly to the control system of the bioreactors. The production of GFP was then measured on-line, the signal was interfaced directly with other controlling parameters, thereby allowing the microbial process to be controlled directly based on recombinant protein expression. A positive expression correlation between on-line and off-line data was obtained. Protein accretion measured off-line was quantified using both LC-MS and plate reader assays. The potential of such a sensor for many aspects of process development is considerable and we have developed a working system which allows the optimisation of production conditions, for example, linking pH control directly to the fusion protein. Results are also presented that illustrate GFP does not alter the cultivation characteristics of the target protein when compared to the native construct. Whether GFP expressed as a fusion influences the solubility of the target protein is also discussed.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	0168-1656 1873-4863
DOI:	10.1016/j.jbiotec.2003.10.039