A simple and rapid method for selecting high producers of recombinant proteins in individual clones of P. pastoris.
The identification of clones expressing high levels of recombinant protein in Pichia pastoris is usually dependant upon SDS-PAGE, Western blotting, or bioactivity-based assays that are labour 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 optimised 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.
Al-Samarrai
Taha H
TH
The Plant and Food Research Institute of New Zealand Ltd, Palmerston North, New Zealand. Tal-Samarrai@hortresearch.co.nz
Kirk
Christopher A
CA
Jones
William T
WT
Harvey
Dawn
D
Templeton
Matthew D
MD
D015153:Blotting, Western; D020650:Combinatorial Chemistry Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D057166:High-Throughput Screening Assays / Q000379:methods*; D008969:Molecular Sequence Data; D010843:Pichia / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins