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Design and production of artificial repeat
proteins with tailored binding sites
Philippe Minard
Modélisation et Ingénierie des Protéines
MIP Lab (IBBMC) Orsay
I B B M C
Part I
INTRODUCTION TO PROTEIN SCAFFOLDS
AND PROTEIN REPEATS
How binding sites appear in natural proteins ?
Biological evolution works like a tinkerer, “who does not know exactly
what he is going to produce, but uses whatever he finds around him
whether it be pieces of string, fragments of wood, or old cardboards;
in short, it works like a ‘bricoleur’ who uses everything at his disposal
to produce some kind of workable object”
Jacob, F. (1977) Evolution and tinkering. Science 196, 1161–1166
The same natural fold is commonly found in different proteins interacting with very
different molecular partners.
 Divergent evolution of binding sites from a single fold is a
common process
Protein scaffolds
Z domain
(Affibodies™)
10FnIII
(Monobodies)
Neocarzinostatin
Lipocalin
(Anticalin®)
Protein repeats as scaffold?
Ki = 5.9 x 10-14 M
Kobe and Deisenhofer . Nature. 1993 Dec 23-30;366(6457):751-6.
Crystal structure of porcine ribonuclease inhibitor, a protein with
leucine-rich repeats
2bnh.pdb
Two « families » of proteins made from repeats
Leucine Rich Repeats
Ankyrin repeats
How to create new specific binding proteins ?
Designed Ankyrin Repeats Proteins : DARPINS
Part II
ALPHA-REP DESIGN
Looking for an optimal protein scaffold ?
Screen PDB :
- Origin thermophilic organism
- Monomeric protein
- Moderate Size (>75, <200)
- No SS bonds
- Functionally versatile
PDB code: 1TE4
• Origin : thermophilic Archae
(Methanobacterium thermoautotrophicum )
Julien, O., Gignac, I., Hutton, A., Yee, A., Arrowsmith,
C. H. & Gagne, S. M. (2006). J Biomol NMR 35, 149-54.
• Many homologs in Archae and
thermophilic bacteria. Other
homologs in eukaryotes
• Unknown function
Procedure for repeat Sequence
definition
The goal is to create a sequence database
of mutually compatible repeats
1 Blast a non redundant sequence database with
the sequence of two folded repeats (C-cap
excluded)
2 Keep only the aligned part of the collected
sequences. Split it in blocks corresponding to
structural repeats
3 Most frequent residue at each position
of multiple
sequence alignment define Consensus 1
GDERA………LGKI
Procedure for repeat Sequence definition (2)
4
Extend repeats collection : Blast non redundant sequence database
with a theoritical « penta-consensus 1 sequence » (GDERA………LGKI)5
GDERA………LGKI GDERA………LGKI
GDERA………LGKI
GDERA………LGKI
GDERA………LGKI
5
Select 100 closest natural sequences, split in 500 repeats. Align all 500 different
repeats.
A consensus with sharp contrast :
Sequence positions in repeat are not equally conserved
Where are the conserved positions?
H-Bond between sides
chains R21 and D15 of
the following repeat
A5,V6,L9,L13,V20,A24,A25,A27,L28
D15,R21
Why Hypervariable residues ?
18,19,21,22,25,30
• Part III
Library construction and selections
+
+
n
To make a repeat Library
1. Synthetize micro-genes corresponding to one repeat,
with randomized hypervariable positions
2
Directional polymerization of micro-genes
ProtectedEnd (N cap)
Ccap
Phage display / Expression phagemid
How to tune side chains randomization ?
0,18
18 nat
0,16
0,14
26 Nat
0,12
0,1
0,08
0,06
0,04
0,02
0
A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
Frequencies of the 20 Aminoacids in two different positions of
natural alphaRep-like repeat family
Step I : Synthetize and amplify microgenes
micro gene = 1 repeat
31 AA total and 6 Variable AA for each repeat
Circular amplification(RCA).
Phi 29 pol
Ob13-14
ligation
BsmBI
BsmBI
Make a circle with
oligos
BsmBI
Close by ligation
BsmBI
BsmBI
Cleave : monomeric microgene pool
(Non symetric cohesive ends)
Library construction : polymerize microgenes
KpnI
BbsI
BspMI
In vector heteropolymerization
+
pHDiex-acc
BspMI
capping with
the N-cap
module
BsmBI
BsmBI BsmBI
BspMI
BsmBI
BspMI
BspMI
pHDiex-aRep
final digestion and
reclosing ligation
aRep library 2.1
Library Design
• Libary diversity : 1.7 109 indépendant clones
• Proportion of correct sequences : 77%. High « Foldability »
• Number of repeat /protein : from 1 to 10 + (average 2.8)
Biophysical properties of purified aRep
with variable sizes and sequences
Far UV Circular Dichroïsm
Size exclusion Chromatography
 aRep are soluble, folded and very stable
Differential Scanning Calorimetry
J. Mol. Biol. (2010) 404, 307–327
Structure aRep
PDB Code 3LTJ
J. Mol. Biol. (2010) 404, 307–327
Collaboration Marc Graille et Herman van Tilbeurgh
Phage display selection of new specific proteins
Monitoring of selections
S
Targets
1
2
3
P
4
Library
Round 1
Round 2
E1
E2
E3
c
uc
c
uc
c
uc
c
uc
Round 3
E1 = specific elution 30’
E2= specific elution 30 to 90’
E3= Acid Elution
• Part III
Specific aREP characterization and
applications
Selection of specific binders from aRep library
Process
-
Three rounds of selection
clonal identification ( Phage Elisa)
Confirmation by Elisa on soluble protein
KD : ITC, SPR….
Results
- Binders identified for >20 non-related soluble proteins
- Kd from low nM to mM
Protein Engineering and Modelling
aRep Binders Characterization
Kd aRep/target
from nM To mM
koff = 1,7. 10- 4 Sec-1
kon= 3.10 4 M-1 Sec-1
Crystal structures of aRep Target complexes
.
With M Graille and H vTilbeurgh
PLoS ONE 2013 8(8): e71512
aREP scaffold from complexes with different targets
Scaffold of aRep
extracted from 5
different structures
Comparison of individual repeats structures
 Reduction of conformational heterogeneity
and stabilisation of target protein
 Provide new surfaces for intermolecular contacts
and promote crystal lattice formation.
aRep as crystallization chaperones
Two proteins that gave no crystal without aRep
Fibronectin binding protein (FNE)
KD = 0,18 µM
Collaboration with Hv Tilbeurgh Lab
Expression in living cells
Epifluorescence images on living cells (HEK)
bGFPA
bGFPC
bGFPD
:
 AlphaReps are expressed and stable in Eucaryotic Cells
ALPHAREP: OUTIL DE RECONNAISSANCE MOLECULAIRE
AlphaRep contrôle
Target recognition in living cells
• Co Expression GFP-Rab6 (Golgi) and aRep-mCherry
CMV
GFP-Rab6
Collaboration Sandrine Moutel , Franck Perez I Curie Paris
Flag
αRep
mcherry
aRep(antiGFP)-mCherry
Target recognition in living cells (2)
Co-expression : GFPs-NLS and antiGFPAlphaReps
bGFPC
bGFPA
bGFPD
AlphaRep contrôle
G
R
M
AlphaRep-mcherry: Red
Localized GFP : Green
ALPHAREP: OUTIL DE RECONNAISSANCE MOLECULAIRE
Epifluorescence and Confocal images
Conclusions
 AlphaREP library: very efficient source of tight binding and
specific proteins
 A generic library : AlphaREP selection needs no immunization
 No SS Bonds, Cysteine free , very stable proteins
=> AlphaREP are efficiently expressed and engineered
 Very promising as crystallisation helpers
 Expressed, stable and functionnal in living cells
Collaborations
FAAM Lab (Orsay)
• Herman van Tilbeurgh
• Ines Gallay
• Marc Graille
(actuellement labo Biochimie de l’ X)
IBPC Paris
• Martin Picard
• Jean Luc Popot, M Zoonens
I .Curie : Franck Perez
LEBS:
Jacqueline Cherfils
Alexis Gautreau
Supports: