Evaluation of the GFP signal and its aptitude for novel on-line monitoring strategies of recombinant fermentation processes.
A high number of economically important recombinant proteins are produced in Escherichia coli based host/vector systems. The major obstacle for improving current processes is a lack of appropriate on-line in situ methods for the monitoring of metabolic burden and critical state variables. Here, a pre-evaluation of the reporter green fluorescent protein (GFP) was undertaken to assess its use as a reporter of stress associated promoter regulation. The investigation of GFP and its blue fluorescent variant BFP was done in model fermentations using E. coli HMS 174(DE3)/pET11 aGFPmut3.1 and E. coli HMS174(DE3)/pET1aBFP host/vector systems cultured in fed-batch and chemostat regime. Our results prove the suitability of the fluorescent reporter proteins for the design of new strategies of on-line bioprocess monitoring. GFPmut3.1 variant can be detected after a short lag-phase of only 10 min, it shows a high fluorescence yield in relation to the amount of reporter protein, a good signal to noise ratio and a low detection limit. The fluorescence-signal and the amount of fluorescent protein, determined by ELISA, showed a close correlation in all fermentations performed. A combination of reporter technology with state of the art sensors helps to develop new strategies for efficient on-line monitoring needed for industrial process optimisation. The development of efficient monitoring will contribute to advanced control of recombinant protein production and accelerate the development of optimised production processes.
Reischer
Helga
H
Institute for Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Muthgasse 18B, A-1190 Wien, Austria.
Schotola
Irene
I
Striedner
Gerald
G
Pötschacher
Florentina
F
Bayer
Karl
K
D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D005285:Fermentation / Q000502:physiology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D009862:Online Systems; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins