Proteolytic selection for protein folding using filamentous bacteriophages.
BACKGROUND
Filamentous bacteriophages have been used for the selection of folded peptide and protein 'ligands' by binding the phage to 'receptor'-coated solid phase. Here, using proteolysis, we have developed a technique for the selection of folded and stable proteins that is independent of their binding activities.


RESULTS
When a 21-residue peptide comprising a protease cleavage site was introduced into the flexible linker between the second and third domains of the minor coat protein p3 of filamentous bacteriophage, the phages could be cleaved by trypsin and were rendered non-infective. By contrast, phages displaying mutant barnases at this site were resistant to proteolysis, but were cleaved and their infectivity was destroyed as the temperature was raised. By mixing phages bearing two barnase mutants of differing stability, and adding protease at a temperature at which one mutant was resistant and the other was sensitive, we were able to enrich by 1.6 x 10(4)-fold for phages bearing the more stable barnase.


CONCLUSIONS
The approach provides a means for the selection of folded and stable proteins, and may be applicable to the selection of de novo proteins.
Kristensen
P
P
MRC, Centre for Protein Engineering, Hills Road, Cambridge, CB2 2QH, UK. pk2@mrc-lmb.cam.ac.uk
Winter
G
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D005822:Genetic Vectors; D006868:Hydrolysis; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017510:Protein Folding; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D008840:Microfilament Proteins; C026615:villin; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease