High Throughput Selection of Stable Protein Variants Using Green - - PowerPoint PPT Presentation

University of Washington Joshua Cho, William Harvey, & Stephen Rettie

High Throughput Selection of Stable Protein Variants

Using Green Fluorescent Protein to Quantify Protein Stability

Engineered Proteins Play Important Roles in Many Fields

Research Superfolder GFP – A more stable GFP. (Pedelacq JD et al, 2006, Nat Biotechnol) Energy & Industry T-PRIMED – Cellulase enzymes with higher thermostability and enzymatic activity. (Trudeau DL, 2014, Biotechnol Bioeng) Drug Delivery Stabilization of nanocages for effective drug delivery. (Ardejani MS et al, 2011, Biochemistry) Therapeutics ZMAPP – Cocktail of Ebola Antibodies (Daniel Murin, The Scripps Research Institute)

Current Methods of Improving Stability are Inefficient

Prediction & Modeling Mutagenesis Cloning Expression Purification Melting Curves

Frac%on ¡of ¡Folded ¡ Protein ¡

Guanidine ¡Concentra%on ¡(M) ¡

GAL1 Promoter GFP

Generalizable High-Throughput

Evaluation and Evolution of Protein Stability

GAL4 Protein of Interest VP1 6

Degro n

Co-Localization of GAL4 & VP16 Activates GFP Expression

CYC1 Promoter

GAL4 VP16 GAL 4 VP16 ¡ GAL1 Promoter GFP

Within PyE1 genome

GAL 4 VP16 ¡ GAL 4 VP16 ¡

Hypothesis: High GFP = High Stability vs Low GFP = Low Stability

GAL1 Promoter GFP

Within PyE1 genome

E3 Ubiquitin Ligase

Stable Unstable

Ub ¡ Ub ¡ Ub ¡

(Folded & Unfolded Protein Figures: Bowman G et al, 2011, J. Am. Chem. Soc.)

Degrons Destabilize Protein Complexes

CYC1 Promoter GAL4 Protein of Interest VP16 Degron GAL 4 VP16 ¡ Protein ¡of ¡Interest ¡ Degron E3 Ubiquitin Ligase

Ub ¡ Ub ¡ Ub ¡ Ub ¡ Ub ¡

Possible Degron Sites Within the Plasmids

Gal4 Protein

Deg1 Deg2 DEGRO N

Hypothesis: Position Affects Degron’s Destabilizing Influence

GAL 4 Protein

• f

Interest VP1 6 GAL4 Protein

• f

Interest VP1 6 Deg GAL 4 Protein

• f

Interest VP1 6 Deg GAL 4 Protein

• f

Interest VP16 Deg GAL4 Protein

• f

Interest VP1 6 Deg

Predicted ¡Most ¡Stable ¡ Deg0: ¡ Deg3: ¡ Deg2: ¡ Deg1: ¡ Deg4: ¡

GAL1 Promoter GFP

Generalizable High-Throughput

Evaluation and Evolution of Protein Stability

GAL4 Protein of Interest VP1 6

Degro n

High Throughput Evaluation Allows for Simultaneous Testing of Millions of Variants

Create Random Mutagenesis Library Through Error-Prone PCR X XX X X XXX X X X

Amplify gene or gene fragment using mutazyme. Purify target fragment containing mutations (X)

Sort cells using Fluorescence Activated Cell Sorting (FACS).

X X

Place into our system by assembling and transforming library into yeast.

Selecting for Stable Variants Using Fluorescence-Activated Cell Sorting (FACS)

Representative FACS Plot

Cell Size GFP Output (AU) Regrow & Re-sort

Evaluation and Evolution of Protein Stability

GAL1 Promoter GFP

Generalizable High-Throughput

GAL4 Protein of Interest VP1 6

Degro n

Data Supports Degron Position Hypothesis

Stability

Expected Actual Deg0 Deg2, Deg3 Deg1, Deg4 Deg1, Deg4 Deg2, Deg3 Deg0

Mean GFP Output (AU) Degron Position

0 ¡ 5000 ¡ 10000 ¡ 15000 ¡ 20000 ¡ 25000 ¡ 30000 ¡ 35000 ¡ 40000 ¡ 45000 ¡

PyE1 Deg0 Deg1 Deg2 Deg3 Deg4

Using BINDI and its Variants in Our System

BINDI – Binds to BHRF1 gene of Epstein-Barr Virus

Image of BINDI (pdb:4OYD), E. Procko, 2014, Cell, Made using PyMOL

BbpD04.3 ¡– ¡Stable ¡variant ¡ BbpD04 ¡– ¡Unstable ¡variant ¡

0 ¡ 0.25 ¡ 0.5 ¡ 0.75 ¡ 1 ¡ 0 ¡ 1 ¡ 2 ¡ 3 ¡ 4 ¡ 5 ¡

Fraction of Folded Protein Guanidine Concentration (M)

Bindi ¡and ¡BbpD04.3 ¡Denature ¡at ¡the ¡Same ¡Concentra%on ¡of ¡Guanidine ¡

Point of Denaturation

BINDI Variants Follow Expected Stability Trend

BbpD04.3 = 3.705 M BINDI = 3.5875 M BbpD04 = 3.088 M

Relative Stabilities Between BINDI Variants Not Preserved

Stability

Expected No Insert BbpD04.3 BINDI PyE1 No Insert BbpD04.3 BINDI BbpD04 BbpD04

Degron ¡Posi%on ¡ Mean GFP Output (AU) PyE1 Deg0 Deg1 Deg2 Deg3 Deg4

Insertion of BINDI Variants Support Degron Position Hypothesis

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 PyE1

PyE1 No Insert BbpD04.3 BINDI BbpD04

Stability

Expected Actual Deg0 Deg2, Deg3 Deg1, Deg4 Deg1, Deg4 Deg2, Deg3 Deg0

Mean GFP Output (AU) Degron ¡Posi%on ¡

0 ¡ 10000 ¡ 20000 ¡ 30000 ¡ 40000 ¡ 50000 ¡ 60000 ¡

PyE1 (Negative Control) BbpD04 Deg2 Clone Library Pre-Sort Library Post-Sort 1 Library Post-Sort 2

Selecting for Stable Variants With FACS

Cell Count GFP Output per Cell (AU)

Ongoing Work

Purify and Express New Protein Variant Confirm Improved Stability Analyze Samples through Flow Cytometry

0 ¡ 10000 ¡ 20000 ¡ 30000 ¡ 40000 ¡ 50000 ¡ 60000 ¡

0 ¡ 0.25 ¡ 0.5 ¡ 0.75 ¡ 1 ¡ 0 ¡ 1 ¡ 2 ¡ 3 ¡ 4 ¡ 5 ¡

Frac%on ¡of ¡Folded ¡Protein ¡ Guanidine ¡Concentra%on ¡(M) ¡

How Could You Use It?

Clone protein sequence of interest into Deg0, Deg1 and Deg2. Perform an error-prone PCR. Run transformed yeast through FACS.

Gal4 Protein Deg1 Deg2 DEGRON

X XX X X XXX X X X

Two New BioBricks Submitted and One Improved

GAL4 VP16

Gal4-Vp16:

VP16 GAL4 Degron

Deg2:

Degron

Degron:

Improved:

BBa_K1408001 BBa_K1408002 BBa_K1408000

Submitted:

GAL4

BBa_K1179014

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

• f 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

University of Washington Joshua Cho, William Harvey, & Stephen Rettie

High Throughput Selection of Stable Protein Variants

Using Green Fluorescent Protein to Quantify Protein Stability