Abstract
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
Combinatorial Chemistry & High Throughput Screening
Title: A Simple and Rapid Method for Selecting High Producers of Recombinant Proteins in Individual Clones of P. pastoris
Volume: 13 Issue: 5
Author(s): Taha H. Al-Samarrai, Christopher A. Kirk, William T. Jones, Dawn Harvey and Matthew D. Templeton
Affiliation:
Keywords: P. pastoris, GFP, GFP-tagged recombinant proteins, GFP fluorescence, Venturia inaequalis, EST 38-GFP, EST40-GFP, EST CIN1-GFP, High throughput screening
Abstract: 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.
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Cite this article as:
H. Al-Samarrai Taha, A. Kirk Christopher, T. Jones William, Harvey Dawn and D. Templeton Matthew, A Simple and Rapid Method for Selecting High Producers of Recombinant Proteins in Individual Clones of P. pastoris, Combinatorial Chemistry & High Throughput Screening 2010; 13 (5) . https://dx.doi.org/10.2174/138620710791292967
DOI https://dx.doi.org/10.2174/138620710791292967 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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