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, allowing expression of both GFP and the target protein as a fusion. Cultivations were carried out at the 20l 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.
Jones
Jo J
JJ
Gene Expression and Protein Biochemistry, GlaxoSmithKline, New Frontiers Science Park, Coldharbour Road, Harlow, Essex, CM19 5AD, UK. Jo_J_Jones@gsk.com
Bridges
Angela M
AM
Fosberry
Andrew P
AP
Gardner
Sharmila
S
Lowers
Robert R
RR
Newby
Rachel R
RR
James
Philip J
PJ
Hall
Richard M
RM
Jenkins
Owen
O
D015374:Biosensing Techniques / Q000295:instrumentation*; D005285:Fermentation; D005822:Genetic Vectors / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis
D011993:Recombinant Fusion Proteins