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Effects of in Situ Cobalt Ion Addition on the Activity of a GFP‐OPH Fusion Protein: The Fermentation Kinetics
Author(s) -
Wu ChiFang,
Valdes James J.,
Bentley William E.
Publication year - 2001
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp010043z
Subject(s) - inducer , cobalt , green fluorescent protein , lac operon , chemistry , fermentation , fusion protein , kinetics , fluorescence , fusion , biochemistry , ion , bioreactor , biophysics , nuclear chemistry , escherichia coli , inorganic chemistry , biology , recombinant dna , organic chemistry , physics , linguistics , philosophy , quantum mechanics , gene
The effects of cobalt ion addition and inducer concentration were studied in the fermentation of E. coli BL21 expressing a GFP (green fluorescent protein)‐OPH (organophosphorus hydrolase) fusion protein. It was found that cobalt ion addition improved the OPH activity significantly. When 2 mM of CoCl 2 was supplied during the IPTG‐induction phase, OPH activity was enhanced ∼10‐fold compared to the case without cobalt or by the addition of cobalt to the cell extracts. Results indicate, therefore, that incorporation of the cobalt during synthesis is needed for enhanced activity. Also, the maximum OPH activity was not linearly related to inducer concentration. A mathematical model was then constructed to simulate these phenomena. Model parameters were determined by constrained least‐squares and optimal IPTG and cobalt addition concentrations were obtained, pinpointing the conditions for the maximum productivity. Finally, the GFP fluorescence intensity was found linear to the OPH activity in each fermentation, demonstrating the function of GFP for monitoring its fusion partner's quantity in the bioreactor.