High Throughput Selection of Stable Protein Variants Using Green Fluorescent Protein to Quantify Protein Stability University of Washington Joshua Cho, William Harvey, & Stephen Rettie
Engineered Proteins Play Important Roles in Many Fields Drug Delivery Research Stabilization of Superfolder GFP – nanocages A more stable GFP. for effective drug delivery. (Pedelacq JD et al, 2006, Nat Biotechnol) (Ardejani MS et al, 2011, Biochemistry) Energy & Industry Therapeutics T-PRIMED – ZMAPP – Cellulase enzymes Cocktail of Ebola with higher Antibodies thermostability and (Daniel Murin, enzymatic activity. The Scripps Research Institute) (Trudeau DL, 2014, Biotechnol Bioeng)
Current Methods of Improving Stability are Inefficient Mutagenesis Prediction & Modeling Cloning Frac%on ¡of ¡Folded ¡ Protein ¡ Guanidine ¡Concentra%on ¡(M) ¡ Melting Curves Purification Expression
Evaluation and Evolution of Protein Stability High-Throughput Generalizable Degro VP1 n Protein of 6 Interest GAL4 GAL1 GFP Promoter
Co-Localization of GAL4 & VP16 Activates GFP Expression GAL4 VP16 CYC1 Promoter VP16 ¡ GAL 4 Within PyE1 GAL1 GFP Promoter genome
Hypothesis: High GFP = High Stability vs Low GFP = Low Stability Stable Unstable Ub ¡ Ub ¡ E3 Ubiquitin Ligase Ub ¡ VP16 ¡ GAL 4 VP16 ¡ GAL 4 Within PyE1 GAL1 Promoter GFP genome (Folded & Unfolded Protein Figures: Bowman G et al, 2011, J. Am. Chem. Soc.)
Degrons Destabilize Protein Complexes E3 Ubiquitin Ligase Ub ¡ Ub ¡ Ub ¡ Ub ¡ Ub ¡ VP16 ¡ CYC1 Degron Protein ¡of ¡Interest ¡ Degron GAL4 Protein of Interest VP16 Promoter GAL 4
Possible Degron Sites Within the Plasmids Gal4 Protein Deg2 Deg1 DEGRO N
Hypothesis: Position Affects Degron’s Destabilizing Influence Predicted ¡Most ¡Stable ¡ Deg0: ¡ Protein VP1 of 6 Interest GAL 4 Protein Deg3: ¡ Deg2: ¡ Protein VP1 of VP16 Deg Interest of 6 Interest GAL Deg GAL 4 4 Deg Protein Deg4: ¡ Protein VP1 Deg1: ¡ Deg of of 6 VP1 Interest Interest 6 GAL4 GAL4
Evaluation and Evolution of Protein Stability High-Throughput Generalizable Degro VP1 n Protein of 6 Interest GAL4 GAL1 GFP Promoter
High Throughput Evaluation Allows for Simultaneous Testing of Millions of Variants Create Random Mutagenesis Library Through Error-Prone PCR X X Amplify gene or gene fragment using mutazyme. Place into our system by X assembling and XX transforming library into X X yeast. X X XXX Sort cells using Fluorescence Activated Cell Sorting (FACS). X Purify target fragment containing mutations (X)
Selecting for Stable Variants Using Fluorescence-Activated Cell Sorting (FACS) Representative FACS Plot Regrow & Re-sort GFP Output (AU) Cell Size
Evaluation and Evolution of Protein Stability High-Throughput Generalizable Degro VP1 n Protein of 6 Interest GAL4 GAL1 GFP Promoter
Data Supports Degron Position Hypothesis 45000 ¡ 40000 ¡ Stability 35000 ¡ Expected Actual Mean GFP Output (AU) 30000 ¡ Deg0 Deg0 25000 ¡ 20000 ¡ Deg2, Deg2, Deg3 Deg3 15000 ¡ Deg1, Deg1, 10000 ¡ Deg4 Deg4 5000 ¡ 0 ¡ PyE1 Deg0 Deg1 Deg2 Deg3 Deg4 Degron Position
Using BINDI and its Variants in Our System BINDI – Binds to BHRF1 gene of Epstein-Barr Virus BbpD04.3 ¡– ¡Stable ¡variant ¡ BbpD04 ¡– ¡Unstable ¡variant ¡ Image of BINDI (pdb:4OYD), E. Procko, 2014, Cell, Made using PyMOL
BINDI Variants Follow Expected Stability Bindi ¡and ¡BbpD04.3 ¡Denature ¡at ¡the ¡Same ¡Concentra%on ¡of ¡Guanidine ¡ Trend 1 ¡ BbpD04.3 = 3.705 M BINDI = 3.5875 M 0.75 ¡ Fraction of Folded Protein BbpD04 = 3.088 M Point of Denaturation 0.5 ¡ 0.25 ¡ 0 ¡ 0 ¡ 1 ¡ 2 ¡ 3 ¡ 4 ¡ 5 ¡ Guanidine Concentration (M)
Relative Stabilities Between BINDI Variants Not Preserved PyE1 No Insert BbpD04.3 Mean GFP Output (AU) BINDI BbpD04 Stability Expected No Insert BbpD04.3 BINDI BbpD04 PyE1 Deg0 Deg1 Deg2 Deg3 Deg4 Degron ¡Posi%on ¡
Insertion of BINDI Variants Support Degron Position Hypothesis 60000 ¡ PyE1 No Insert 50000 ¡ BbpD04.3 BINDI BbpD04 40000 ¡ Mean GFP Output (AU) Stability 30000 ¡ Expected Actual Deg0 Deg0 20000 ¡ Deg2, Deg2, 10000 ¡ Deg3 Deg3 Deg1, Deg1, 0 ¡ Deg4 Deg4 PyE1 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 Degron ¡Posi%on ¡
Selecting for Stable Variants With FACS PyE1 (Negative Control) BbpD04 Deg2 Clone Library Pre-Sort Library Post-Sort 1 Library Post-Sort 2 Cell Count GFP Output per Cell (AU)
Ongoing Work Purify and Express New Analyze Samples through Confirm Improved Stability Protein Variant Flow Cytometry 60000 ¡ 50000 ¡ Frac%on ¡of ¡Folded ¡Protein ¡ 1 ¡ 40000 ¡ 0.75 ¡ 30000 ¡ 0.5 ¡ 20000 ¡ 0.25 ¡ 10000 ¡ 0 ¡ 0 ¡ 0 ¡ 1 ¡ 2 ¡ 3 ¡ 4 ¡ 5 ¡ Guanidine ¡Concentra%on ¡(M) ¡
How Could You Use It? Clone protein Run transformed Perform an error-prone PCR. sequence of interest yeast through into Deg0, Deg1 and FACS. Deg2. Gal4 Protein Deg2 Deg1 DEGRON X XX X X X X XXX X
Two New BioBricks Submitted and One Improved Improved: BBa_K1408001 BBa_K1179014 Gal4-Vp16: GAL4 GAL4 VP16 Submitted: Deg2: BBa_K1408002 GAL4 Degron VP16 BBa_K1408000 Degron: Degron
Spreading the Joys of Fluorescent Proteins Bennett Elementary, UW Engineering Discovery Days
A Huge Thanks To • Stan Fields, UW Genome Sciences – Ben Jester • David Baker, UW Biochemistry – Eric Procko • Eric Klavins, UW Electrical Engineering • UW Departments: Bioengineering, Biochemistry, Biology, Microbiology, College of Engineering • Students: – Edward Chang, Andrew Chau, Joshua Cho, Chris Choe, William Harvey, Alex Kang, Julia Lim, Harman Malhi, Colton McDavid, Krista Nguyen, Anastasia Nicolov, Ahmed Qureshi, Stephen Rettie • Advisors: – Nick Bolten, Cassie Bryan, Arjun Khakhar, Robert Lamm, Erik Murphy, Rashmi Ravichandran, David Younger
High Throughput Selection of Stable Protein Variants Using Green Fluorescent Protein to Quantify Protein Stability University of Washington Joshua Cho, William Harvey, & Stephen Rettie
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