Abstract
Avian reovirus capsid protein σB was genetically fused with a histidine (His 6 ) tag and a UV-optimized green fluorescent protein (GFPuv) and expressed in Sf-9 cells. The fluorescence of GFPuv allowed for easy identification of protein localization and revealed that the fusion protein was quite stable in the cell culture. The fluorescence intensity (FI) exhibited a linear relationship (r 2 = 0.93) with the recombinant protein yield and therefore allowed for on-line tracking of the expression profile, which revealed an extremely high maximum yield of 70 μg per 10 6 cells. The recombinant protein was purified via immobilized metal affinity chromatography (IMAC) and a high purity (85%) was achieved in one step. During the purification, the fluorescence again enabled qualitative and quantitative monitoring of when and how much the desired product was eluted. The GFP-tagging strategy eliminated the need for cumbersome and time-consuming assays (e.g. Western blot or ELISA) for product analysis, thus GFP is an effective non-invasive on-line marker for the expression and purification of recombinant proteins in the baculovirus expression system. © 2004 Elsevier Inc. All rights reserved.