Toward development of a screen to identify randomly encoded, foldable sequences.

Area of Science:

• Protein folding and structural biology
• Biotechnology and molecular engineering
• Yeast secretory pathway applications

Background:

• Identifying stable protein structures from random sequences is crucial for protein folding research.
• The yeast secretory pathway's quality control system can be leveraged to select for folded proteins.
• Previous methods identified stabilizing mutations but were not broadly applicable to random libraries.

Purpose of the Study:

• To develop and test a novel multistep screening strategy for identifying foldable sequences within random polypeptide libraries.
• To combine yeast secretion quality control with generic tag-based immunodetection for broad applicability.
• To assess the efficacy of the method in isolating sequences with enhanced thermal stability.

Main Methods:

• Screening a random polypeptide library encoding mutations in a bovine pancreatic trypsin inhibitor variant with a generic tag.
• Employing an initial on-plate screen followed by liquid-culture secretion and gel electrophoresis assays to eliminate false positives.
• Utilizing yeast's inherent protein folding quality control mechanisms.

Main Results:

• Initial screening yielded numerous false positives (secreted but not foldable sequences).
• Subsequent screening steps successfully excluded non-foldable sequences.
• Three positive clones were isolated, exhibiting midpoint thermal denaturation temperatures 10-16°C higher than the wild-type variant.

Conclusions:

• The developed multistep screening method effectively identifies novel, stably folded polypeptide sequences from random libraries.
• This approach offers a valuable tool for protein engineering and understanding protein folding principles.
• The method demonstrates potential for discovering sequences with significantly improved thermal stability.