Colony filtration blotting for screening soluble expression in Escherichia coli.
We have developed a screen for detecting E. coli colonies that produce soluble recombinant target proteins at the colony level: the colony filtration (CoFi) blot. Colonies are transferred, induced and lysed on a filter membrane that can separate soluble proteins from inclusion bodies. Upon lysis, the soluble proteins diffuse through the filter membrane and are captured on a nitrocellulose membrane. The nitrocellulose membrane is incubated with antibodies or probes specific for the target protein and are then developed. In the resulting image, colonies expressing soluble protein can easily be identified. This protocol can be used to screen thousands of constructs in a matter of days, making it very suitable for expression libraries. The protocol is robust and flexible with regard to lysis conditions, induction temperatures and strains. The method requires only standard laboratory equipment and is based on immunochemicals used for western blotting. The following protocol describes the screening of a DNA library with detection done using chemiluminescence. Depending on induction temperature, the whole procedure can be performed in <2 d.
Dahlroth
Sue-Li
SL
Department of Biochemistry and Biophysics, Stockholm University, S-106 91 Stockholm, Sweden.
Nordlund
Pär
P
Cornvik
Tobias
T
D003101:Collodion; D004926:Escherichia coli / Q000235:genetics*; D005374:Filtration / Q000379:methods; D015151:Immunoblotting; D015336:Molecular Probe Techniques; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D011993:Recombinant Fusion Proteins; D003101:Collodion