Member # 1
posted 16. May 2002 12:24
Proc. Natl. Acad. Sci. USA, 0.1073/pnas.102172099
Toward development of a screen to identify randomly encoded, foldable sequences
by Yoshihisa Hagihara and Peter S. Kim
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°C higher than the original bovine pancreatic trypsin inhibitor variant. Thus, this multistep screening method may be useful for finding novel, foldable sequences.
[ 16 May 2002, 12:24: Message edited by: Moderator ]