title: Evaluation of the GFP signal and its aptitude for novel on-line monitoring strategies of recombinant fermentation processes.
abstract: 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.
authors.affiliation: Institute for Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Muthgasse 18B, A-1190 Wien, Austria.
authors.forename: Helga
authors.initials: H
authors.lastname: Reischer
authors.forename: Irene
authors.initials: I
authors.lastname: Schotola
authors.forename: Gerald
authors.initials: G
authors.lastname: Striedner
authors.forename: Florentina
authors.initials: F
authors.lastname: Pötschacher
authors.forename: Karl
authors.initials: K
authors.lastname: Bayer
mesh_terms: 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*
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins