Use of the green fluorescent protein variant (YFP) to monitor MetArg humanproinsulin production in Escherichia coli

Green fluorescent protein (GFP), a relatively new reporter gene, is making an impact on many aspects of science. The attributes of GFP could also be a pplied to the area of recombinant protein production. The work described he re represents the first experiments using GFP as a tool to monitor recombin ant protein production in real time in the fermentation process. We have co nstructed plasmids containing an operon fusion of the gene encoding MetArg- human proinsulin and reporter gene GFP (GFP, BFP, and YFP variants). The Me tArg-proinsulin and GFP variant reporter protein were overexpressed in Esch erichia coli BL21(DE3) after isopropyl beta-D-thiogalactoside induction. Th e MetArg-proinsulin to YFP protein ratio did not change in the cells during the bioprocess. Since there is a quantitative relationship between the lev el of MetArg-proinsulin concentration and YFP fluorescence, it is possible to measure only YFP fluorescence in order to monitor the production of MetA rg-proinsulin during the bioprocess. The expression level of MetArg-proinsu lin could reach 20-25%. Some 140 mg recombinant MetArg-human proinsulin cou ld be obtained easily from 1 liter of fermentation medium. The MetArg-proin sulin could simply be changed into human insulin by trypsin and carboxypept idase B treatment in later steps. These experiments provide possibilities f or using the YFP reporter gene as a convenient tool to monitor protein expr ession in biotechnological processes. The proposed technique could reduce t he time- and labor-intensive analysis of protein production and would impro ve the efficiency of process development. (C) 1999 Academic Press.