Global Protein Stability
Rx Biosciences offers Global protein stability service using yeast surface display technology to analyze and engineer the stability of large sets of proteins (a “global” analysis). Starting from the in-silico designing of engineered peptide sequences, genes are synthesized, cloned in pRxYD 3.0 yeast display vector and simultaneously expressed at the surface of yeast cells.
The peptides are exposed to different instability agents such as trypsin and chymotrypsin proteases, temperature, pH and chemicals such as urea and guanidinium hydrochloride. High-throughput screening and analysis identifies protein variants with enhanced stability across a wide range of proteins within a single experiment. By identifying protein variants with improved stability through yeast display, we can engineer proteins with enhanced functional properties for various applications, like therapeutic antibodies or industrial enzymes.
Service Highlights
- Optimized yeast display vector.
- High-throughput screening.
- Eukaryotic environment.
- Direct analysis on cell surface.
- Highly applicable in protein engineering.
- All services under one roof.
Experimental Steps
(A) Each yeast cell displays many copies of one test protein fused to Aga2. The c-terminal c-Myc tag is labeled with a fluorescent antibody. Protease cleavage of the test protein (or other cleavage) leads to loss of the tag and loss of fluorescence. (B) Libraries of 104 unique sequences are sorted by flow cytometry. Most cells show high protein expression (measured by fluorescence) before proteolysis (blue). Only some cells retain fluorescence after proteolysis; those above a threshold (shaded green region) are collected for deep sequencing analysis.
(C) Sequential sorting at increasing protease concentrations separates proteins by stability. Each sequence in a library of 19,726 proteins is shown as a gray line tracking the change in population fraction (enrichment) of that sequence, normalized to each sequence’s population in the starting (pre-selection) library. Enrichment traces for seven proteins at different stability levels are highlighted in color. (D) EC50s for the seven highlighted proteins in (C) are plotted on top of the overall density of the 46,187 highest-confidence EC50 measurements from design rounds 1–4. (E) Same data as at left, showing that stability scores (EC50 values corrected for intrinsic proteolysis rates) correlate better than raw EC50s between the proteases.
(F–I) Stability scores measured in high-throughput correlate with individual folding stability measurements for mutants of four small proteins. The wild-type sequence in each set is highlighted as a red circle. (F) Pin1 ΔGunf data at 40°C by thermal denaturation (G) hYAP65 Tm data from (H) Villin HP35 ΔGunf data at 25°C by urea denaturation (I) BBL ΔGunf data at 10°C from by thermal denaturation (Science (2017) 357 (6347):168–175).
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