title: Toward development of a screen to identify randomly encoded, foldable sequences.
abstract: The ability to identify sequences in a randomly encoded polypeptide library that are capable of acquiring unique and stably folded structures would be valuable in the examination of protein-folding issues. The quality control system of the yeast secretory pathway prevents the release of incompletely folded polypeptides. Earlier work has shown that this feature can be used in a screen to identify mutations that increase the stability of a protein. We sought to extend this strategy for use with random sequence libraries by combining a quality-control system-based screen with generic tag-based immunodetection that can be applied to any sequence. To test this method, we screened a library encoding random mutations in a bovine pancreatic trypsin inhibitor variant containing a small generic tag. Initial on-plate screening resulted in a large number of false positives: sequences that were secreted but not foldable. These false positives were excluded successfully in additional screening steps that used a liquid-culture secretion screen and a gel electrophoresis assay. Three positive clones were obtained that showed midpoint thermal denaturation temperatures 10-16 degrees C higher than the original bovine pancreatic trypsin inhibitor variant. Thus, this multistep screening method may be useful for finding novel, foldable sequences.
authors: Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142, USA. hagihara-kappael@aist.go.jp
authors: Yoshihisa
authors: Y
authors: Hagihara
authors: Peter S
authors: PS
authors: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000235:genetics*; D002417:Cattle; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D010455:Peptides / Q000235:genetics*; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D014361:Trypsin Inhibitors / Q000235:genetics*
chemical_list: D019151:Peptide Library; D010455:Peptides; D014361:Trypsin Inhibitors; D007611:Aprotinin