Use of the green fluorescent protein variant (YFP) to monitor MetArg human proinsulin 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 applied to the area of recombinant protein production. The work described here represents the first experiments using GFP as a tool to monitor recombinant protein production in real time in the fermentation process. We have constructed plasmids containing an operon fusion of the gene encoding MetArg-human proinsulin and reporter gene GFP (GFP, BFP, and YFP variants). The MetArg-proinsulin and GFP variant reporter protein were overexpressed in Escherichia coli BL21(DE3) after isopropyl beta-d-thiogalactoside induction. The MetArg-proinsulin to YFP protein ratio did not change in the cells during the bioprocess. Since there is a quantitative relationship between the level of MetArg-proinsulin concentration and YFP fluorescence, it is possible to measure only YFP fluorescence in order to monitor the production of MetArg-proinsulin during the bioprocess. The expression level of MetArg-proinsulin could reach 20-25%. Some 140 mg recombinant MetArg-human proinsulin could be obtained easily from 1 liter of fermentation medium. The MetArg-proinsulin could simply be changed into human insulin by trypsin and carboxypeptidase B treatment in later steps. These experiments provide possibilities for using the YFP reporter gene as a convenient tool to monitor protein expression in biotechnological processes. The proposed technique could reduce the time- and labor-intensive analysis of protein production and would improve the efficiency of process development.
Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, Gdańsk, 80-952, Poland.
S
S
Daabrowski
A
A
Brillowska
J
J
Kur
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010957:Plasmids; D011384:Proinsulin / Q000737:chemistry / Q000235:genetics*
D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011384:Proinsulin; D001120:Arginine; D008715:Methionine