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Green fluorescent protein (GFP) can be used as markers in numerous bioscience applications due to its visible fluorescence. Availability of a reliable and affordable GFP quantitation device is important for these applications. GFP can be quantitated with a spectrofluorometer, but the accuracy of spectrofluorometric method is affected by the present of biomass and it cannot be used to differentiate the nativity of a GFP. Therefore, one of the objectives of this study was to develop a GFP quantitation method using gel-based imaging analysis. The precision, detection limit, linearity, reproducibility and accuracy of this quantitation method were investigated to demonstrate its reliability and suitability for practical application. Besides, the sensitivity and GFP nativity differentiation test were performed and compared with those obtained with a spectrofluorometer. The cultivation conditions for the GFP production in Escherichia coli BL21(DE3) were optimized using statistical experimental designs. The process variables include agitation rate, temperature, pH of medium, inducer concentration, induction time and inoculum density were screened using the fractional factorial design. The screening step identified that agitation rate, temperature and cell density for protein expression have significant effects on GFP production. Hence, these process variables were further optimized using the Box-Behnken design. The optimal cultivation conditions were 206 rpm agitation rate at 31°C and cell density (OD600 nm) of 1.04 for protein expression. The predicted GFP yield (0.234 g/L) is corresponded well with that obtained experimentally (0.241 g/L). The GFP yield achieved under the optimized conditions is 9-folds higher than that of the unoptimized conditions (0.025 g/L). A similar protein yield was achieved when these optimized conditions were applied in a 2-liter stirred tank bioreactor fermentation. Cell disruption is an important step in intracellular protein purification. Howe

Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%.

The present specification discloses an integrated purification technique for purifying recombinant intracellular protein (GFP) from intact E. coli cells using preparative PAGE column. Under the influence of electric fields, the negatively charged GFP was drifted out from cells without the need of cell disruption process. The electrophoresed cells were observed under SEM and some on the membrane of cells were observed. GFP was then separated from other host cells host protein along polyacrylamide gel and finally eluted electrophoretically from the gel. The invention has successfully recovered the GFP with purity and yeild 95% and 82%, respectively in 100 min operation.