Abstract:
: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.
journal_name
Comb Chem High Throughput Screenjournal_title
Combinatorial chemistry & high throughput screeningauthors
Al-Samarrai TH,Kirk CA,Jones WT,Harvey D,Templeton MDdoi
10.2174/138620710791292967subject
Has Abstractpub_date
2010-06-01 00:00:00pages
377-82issue
5eissn
1386-2073issn
1875-5402pii
BSP/CCHTS/E-Pub/00066journal_volume
13pub_type
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