Cell-free expression of a GFP fusion protein allows quantitation in vitro and in vivo

The green fluorescent protein (GFP) of Aequorea victoria is frequently fused to other proteins to serve as a reporter for gene expression or the localization of proteins in vivo. We report that when a GFP fusion protein is translated in vitro under standard conditions, the GFP portion folds efficiently and becomes fluorescent. This provides a convenient method for monitoring in vitro translation efficiency of a fusion protein, and to screen for improved mutants of GFP. In addition, quantitation of the translation product combined with fluorescence microscopy of the product immunoprecipitated onto beads allows the determination of the density of the fusion protein in microscopic images. A fusion of the tobacco mosaic virus 30 kDa movement protein [1] to the amino terminus of the S65T mutant of GFP [2] was translated using a standard rabbit reticulocyte lysate system incorporating 35S-labeled methionine of known specific radioactivity. The products were electrophoresed using SDS and polyacrylamide gel electrophoresis (SDS–PAGE) followed by autoradiography (Fig. 1a, lane 1). The major product exhibited the expected mobility; minor products of greater mobility are presumed to arise from internal initiation or premature termination. Scintillation counting, combined with knowledge of the number of methionine residues per molecule of the fusion protein, showed that each microliter of the translation reaction contained 3.8 × 10–15 moles of fusion protein. The GFP part of the fusion protein folded properly, as shown by its fluorescence emission spectrum (Fig. 1b). A clear signal was readily detected in as little as 2 ml of the translation reaction diluted 100-fold, corresponding to a fusion protein concentration of approximately 4 × 10–11 M. The fusion protein was immunoprecipitated onto protein G–sepharose beads using an anti-GFP antibody. Only the major translation product and a minor product, presumed to arise from internal initiation 20 aminoacids downstream from the amino Magazine R207