Limitations of yeast surface display in engineering proteins of high thermostability.
Engineering proteins that can fold to unique structures remains a challenge. Protein stability has previously been engineered via the observed correlation between thermal stability and eukaryotic secretion level. To explore the limits of an expression-based approach, variants of the highly thermostable three-helix bundle protein alpha3D were studied using yeast surface display. A library of alpha3D mutants was created to explore the possible correlation of protein stability and fold with expression level. Five efficiently expressed mutants were then purified and further studied biochemically. Despite their differences in stability, most mutants expressed at levels comparable with that of wild-type alpha3D. Two other related sequences (alpha3A and alpha3B) that form collapsed, stable molten globules but lack a uniquely folded structure were similarly expressed at high levels by yeast display. Together these observations suggest that the quality control system in yeast is unable to discriminate between well-folded proteins of high stability and molten globules. The present study, therefore, suggests that an optimization of the surface display efficiency on yeast may yield proteins that are thermally and chemically stable yet are poorly folded.
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
Sheldon
S
Park
Yao
Y
Xu
Xiaoran Fu
XF
Stowell
Feng
F
Gai
Jeffery G
JG
Saven
Eric T
ET
Boder
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D006358:Hot Temperature; D008969:Molecular Sequence Data; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D015003:Yeasts
D019151:Peptide Library; D011506:Proteins; C119365:alpha3D protein, synthetic