The identification of clones expressing high levels of recombinant protein in Pichia pastoris is usually dependent upon SDS-PAGE, Western blotting, or bioactivity-based assays that are labor and time-consuming. We describe a rapid method that images green fluorescence protein (GFP) of individual P. pastoris clones transformed with vectors that express the proteins as GFP C- terminal fusion. In this report we have used the system to monitor expression of three proteins from Venturia inaequalis. Culture plates containing individual colonies were imaged on a Fuji LAS-3000 system and the intensity of fluorescence of GFP [Mean Gray Value (MGV)] of each colony recorded. Two common variables, the time course of expression and induction temperature were also optimized using this method. The results show that colonies with high levels of GFP fluorescence can be successfully used to identify, at an early stage, colonies expressing high levels of recombinant proteins. This correlation can be used to monitor the conditions for optimization of the expression and accumulation of extracellular recombinant protein in medium and to identify fractions containing GFP-tagged recombinant proteins during protein purification.
Keywords: P. pastoris, GFP, GFP-tagged recombinant proteins, GFP fluorescence, Venturia inaequalis, EST 38-GFP, EST40-GFP, EST CIN1-GFP, High throughput screening
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