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

Hagihara Y,Kim PS

doi

10.1073/pnas.102172099

keywords:

subject

Has Abstract

pub_date

2002-05-14 00:00:00

pages

6619-24

issue

10

eissn

0027-8424

issn

1091-6490

pii

102172099

journal_volume

99

pub_type

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