High Throughput Selection of Stable Protein...
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University of Washington Joshua Cho, William Harvey, & Stephen Rettie High Throughput Selection of Stable Protein Variants Using Green Fluorescent Protein to Quantify Protein Stability
Transcript of High Throughput Selection of Stable Protein...
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 Folde
d Protein
Guanidine Concentra%on (M)
GAL1 Promoter GFP
Generalizable High-Throughput
Evaluation and Evolution of Protein Stability
GAL4
Protein of Interest
VP16
Degron
Co-Localization of GAL4 & VP16 Activates GFP Expression
CYC1 Promoter GAL4 VP16
GAL4
VP16
GAL1 Promoter GFP Within PyE1
genome
GAL4
VP16
GAL4 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
GAL4
VP16 Protein of Interest Degron
E3 Ubiquitin Ligase
Ub Ub
Ub
Ub
Ub
Possible Degron Sites Within the Plasmids Gal4 Protein
Deg1 Deg2
DEGRON
Hypothesis: Position Affects Degron’s Destabilizing Influence
GAL4
Protein of
Interest
VP16
GAL4
Protein of
Interest
VP16 Deg
GAL4
Protein of
Interest
VP16
Deg
GAL4
Protein of
Interest VP16
Deg
GAL4
Protein of
Interest VP16
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
VP16
Degron
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 G
FP O
utpu
t (A
U)
Regrow
& R
e-sort
Evaluation and Evolution of Protein Stability
GAL1 Promoter GFP
Generalizable High-Throughput
GAL4
Protein of Interest
VP16
Degron
Data Supports Degron Position Hypothesis
Stability Expected Actual
Deg0
Deg2, Deg3
Deg1, Deg4
Deg1, Deg4
Deg2, Deg3
Deg0
Mea
n G
FP O
utpu
t (A
U)
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
Frac
tion
of F
olde
d Pr
otei
n
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
Mea
n G
FP O
utpu
t (A
U)
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
Mea
n G
FP O
utpu
t (A
U)
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 C
ell C
ount
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 Folde
d 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 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
University of Washington Joshua Cho, William Harvey, & Stephen Rettie
High Throughput Selection of Stable
Protein Variants Using Green Fluorescent Protein to Quantify Protein Stability
