Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli

Abstract

The implementation of efficient technologies for the production of recombinant mammalian proteins remains an outstanding challenge in many structural and functional genomics programs. We have developed a new method for rapid identification of soluble protein expression in E. coli, based on a separation of soluble protein from inclusion bodies by a filtration step at the colony level. The colony filtration (CoFi) blot is very well suited to screen libraries, and in the present work we used it to screen a deletion mutagenesis library.

Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell

“…We selected a set of nine proteins with a midpoint of unfolding (melting temperature; T m ) of the purified protein ranging from 40°C to 68°C, which were cloned into expression plasmids, transformed into E. coli and plated. The colonies were then transferred to a filter membrane that previously has been shown to let soluble proteins pass through while retaining aggregates 26 . After expression, the colonies were incubated at a wide range of temperatures after which they were subjected to concurrent lysis and CoFi 26 (Fig.…”

“…The colonies were then transferred to a filter membrane that previously has been shown to let soluble proteins pass through while retaining aggregates 26 . After expression, the colonies were incubated at a wide range of temperatures after which they were subjected to concurrent lysis and CoFi 26 (Fig. 1a,b).…”

“…The T cagg was determined following a modified protocol based on the CoFi blot 26,46 . Rosetta2 cells (Novagen) were transformed with the expression plasmids, harbouring the gene of interest fused with a C-terminal His 6 -tag, and the cells were plated.…”

“…The method is an extension and modification of our previously published colony filtration (CoFi) blot method 26 . It circumvents most of the problems linked to current stability selection methods while expanding its applicability to virtually any protein target that can be expressed recombinantly in a colony-forming microorganism.…”

Screening of Bacterial Recombinants: Strategies and Preventing False Positives

“…These include strategies like co-expression of chaperone proteins such as GroES, GroEL, DnaK and DnaJ lowering incubation temperature, use of weak promoters, addition of sucrose and betaine in a growth media, use of a richer media with phosphate buffer such as terrific broth (TB), use of signal sequence to export the protein to the periplasmic fraction, fermentation at extreme pH's and use of fusion tags to aid in expression and protein purification (De Marco et al, 2004). A colony filtration (CoFi) blot method for rapid identification of soluble protein expression in E. coli, based on a separation of soluble protein from inclusion bodies by a filtration step at the colony level is described to screen a deletion mutagenesis library by Cornvik et al, (2005) while Coleman et al (2004) report a fluorescent based screening of soluble protein expression where specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. Fusion tags have become indispensable tools for structural and functional proteomics initiatives .…”