Effects of in situ cobalt ion addition on the activity of a gfp-oph fusion protein: the fermentation kinetics.
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 approximately 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.
Wu
C F
CF
Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742, and U.S. Army, ECBC, SBCCOM, Aberdeen Proving Ground, Maryland 21010.
Valdes
J J
JJ
Bentley
W E
WE
D043303:Aryldialkylphosphatase; D003035:Cobalt / Q000494:pharmacology*; D003198:Computer Simulation; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D004950:Esterases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D049452:Green Fluorescent Proteins; D007477:Ions; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008164:Luminescent Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D007477:Ions; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007544:Isopropyl Thiogalactoside; D003035:Cobalt; D004950:Esterases; D043303:Aryldialkylphosphatase