Emission
615 nm
biotinylated
Anti-phage
Streptavidin-
coated
Donor BeadsAnti-M13
conjugated
Acceptor Beads
1O2
Excitation
680 nm
T7-expressed constructs can be specifically
phosphorylated by Akt1
This phosphorylation can be detected by anti-
phospho-consensus antibodies
T7-expressed constructs can be specifically
phosphorylated by c–Src
This phosphorylation can be detected by anti-
phosphotyrosine antibody-coupled beads
Constructs expressed at the surface of M13 can be
specifically phosphorylated
c-Src phage phosphorylation can be monitored with
anti-phospho tyrosine beads, even in amplified cultures
Purified M13 phosphorylation can also be detected by
PhosphoSensor beads
Emission
520-620 nm
PhosphoSensor®
(Lewis Metal Chelate)
Acceptor Beads
biotinylated
Anti-phage
Streptavidin-coated
Donor Beads
Excitation
680 nm
LM3+
LM
3+
LM
3+
LM3+
Emission
615 nm
biotinylated
Anti-T7
Streptavidin-
coated
Donor BeadsAnti-T7
conjugated
Acceptor Beads
Excitation
680 nm
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 400
100,000
200,000
300,000
400,000
500,000
600,000
700,000
800,000
900,000
1,000,000
1,100,000
1,200,000
Phage samples processed (triplicats)
Alp
ha
LIS
A c
ou
nts
(c
ps
)
0
25,000
50,000
75,000
100,000
125,000
150,000
175,000
103 104 105 106 1070
pfu
Alp
ha
LIS
A c
ou
nts
(c
ps
)
Standard curve
with T7 library
biotinylated
Anti-T7
Mouse IgG
Streptavidin-coated
Donor Beads
Anti-rabbit IgG conjugated
Acceptor BeadsAnti-RXRXXpS/pT
Rabbit IgG
biotinylated
Anti-T7
Streptavidin-coated
Donor Beads
Anti-phospho tyrosine
conjugated
Acceptor BeadsPhage Titering
E. Coli bacteria were grown overnight and diluted 10-fold according to
Manufacturer’s Instructions. T7 phages (10-3 T7 Phage display, Novagen)
were serial-diluted 10-fold, mixed with molten top agarose, poured on LB-
Agar petri dishes and incubated overnight at 23C. M13 (Ph.D. M13 Phage
Display, New England Biolabs) were processed similarly but X-gal and
IPTG (Sigma-Aldrich) was added to the top agarose and incubation was
done at 37 C. Blue M13 colonies or T7 lysis plaques were counted
manually.
AlphaLISA titering
In a 384 well plate (PerkinElmer), phage preparations were incubated at
23°C with 3 nM biotinylated antibody (Anti-filamentous phage, Sigma-
Aldrich; Novagen) and 20 µg/mL Acceptor beads (Anti-M13 GE Healthcare,
PerkinElmer; Anti-T7 Novagen, PerkinElmer), before addition of
streptavidin-coated Donor beads (PerkinElmer) for 30 min and subsequent
reading on EnVision® multiplate reader(PerkinElmer).
Kinase assays
Normalized phage preparations were incubated at 23°C for 2h with 1 nM
enzyme (c-Src CarnaBio USA; Akt1 Millipore) and 10 µM ATP in a tris-
based buffer containing 10 mM MgCl2. 20 µg/mL Acceptor beads and 3 nM
antibody (Anti-phospho Akt consensus Cell Signaling) were added 1 h prior
to Donor beads. After a final 30 min incubation, plates were read. In some
cases, an additional reading was performed overnight.
PerkinElmer Life and Analytical Sciences, 940 Winter Street, Waltham, MA USA (800) 762-4000 (+1) 203 925-4602 www.perkinelmer.com
Bacteriophage-Expressed Soluble Substrates for Protein Kinases
Mathieu Arcand, Alexandre Marcil, Sophie Dahan, Francesco Lipari, Philippe Roby, Lucille Beaudet and Martina Bielefeld-Sévigny.
PerkinElmer Inc., Montreal, Quebec, Canada, H3J 1R4
Abstract1
Phage-Expressed Proof-of-
Concept Constructs5
c-Src T7 Phage Substrates9
2
M13 Phage Titering6
Akt1 T7 Phage Substrates10
7
Summary11
The human kinome comprises nearly 520 protein kinases that serve as key
mediators of processes such as growth, metabolism, inflammation, cell
division and apoptosis. Because kinases play crucial regulatory roles in so
many processes, they are often linked to disease and represent attractive
pharmacological targets. Despite extensive efforts, there remain numerous
“orphan” kinases for which no substrate has yet been identified. Our goal is to
develop a screening assay for the identification of new substrates for protein
kinases.
We turned towards available M13 and T7 phage display systems since these
bacteriophages exhibit fast and simple replication, and can be used as
expression systems that physically link a protein target with its DNA coding
sequence. We therefore constructed various M13 and T7 phages harbouring
consensus phosphorylation sequences for the tyrosine kinase c-Src and the
serine/threonine kinase Akt1. Normalized phage populations were used as
substrates in kinase assays performed in well plates. The normalization of
phage numbers was carried out using AlphaLISA® beads coupled to
antibodies directed against bacteriophage structural proteins. This method is
an effective substitute for the tedious, yet conventional phage titration.
In a similar bead assay setting, anti-phosphorylated consensus antibodies
were used to determine the phosphorylation of phage-expressed substrates
following kinase assays. We found the substrate constructs expressed at the
surface of M13 and T7 bacteriophages to be phosphorylated by the kinases,
whereas non-phosphorylatable mutants were not. These data suggest that
phosphorylation occurs specifically on the expressed constructs and not on
bacteriophage structural proteins.
Such tailored phages represent a simple, fast, self-replicative and inexpensive
way of determining the kinase activity. Ultimately, this assay platform should
enable us, not only to measure phosphotransferase activity, but also to identify
new targets for virtually any protein kinase.
Program
#1946
Phage Display Systems3
PHOSPHAGE
Phos-
phate
Libray
cDNA
pVIII
pIII
PHOSPHAGE
Libray
peptide
Phos-
phate
pVIII
pIII
PhD PHAGE DISPLAY
Libray
peptide
KINASE
+ATP
pVIII
pIII
M13 BACTERIOPHAGE
6 nm width
990 nm length
Circular genome
5 kB ssDNA
Secreted phages
T7 BACTERIOPHAGE
150 nm sherical
Linear genome
37 kB dsDNA
Lytic phages
NEB
Random
peptide
library
Libray
cDNA
Novagen
Tissue-
based
cDNA
libraries
Materials and Methods4
Akt1 R-X-R-X-X-S/T
Akt SA
RKRNRNKSVEG
RKRNRNKAVEG
c-Src E/D-E/D-X-X-Y-G/W
Src YF KIEEPLFWMFG
Kinase/ Consensus
wt substrate construct
Phosphorylation site
mutant
Akt wtKIEEPLYWMFGSrc wt
518 Protein kinases
~300 orphan kinases
without any
known substrate
~ 90% Ser/Thr kinases
ORPHAN
KINASE
UNKNOWN
SUBSTRATE
??
Because they physically link
protein content to DNA sequence, bacteriophages
could be used as substrates for
protein kinases in a deorphanization strategy
Project Goals
M13 T7
0
100
200
300
400
500
600
700
800
900Src wt
Src YF
103 104 105 106 107 1080
pfu
Alp
ha
Sc
ree
n c
ou
nts
(c
ps
)
T7 Phage Titering8
M13 can easily
be titrated by
AlphaLISA
Many antibody
combinations
work efficiently
108
109
1010
1011
Ph
ag
e d
ilu
tio
n
AMPLIFIED PHAGES PURIFIED PHAGESConventional titering
Grow bacteria overnight
Infect bacteria
Plate on soft agarose
Grow overnight
Count colonies, manually
Dispense in wells
Add beads
Incubate 1h
Read plate
AlphaLISA titering
Acceptor 2
Acceptor 1
Acceptor 4
Acceptor 3
Acceptor 6
Acceptor 5
Acceptor 8
Acceptor 7
c-Src M13 Phage Substrates
Anti-phospho tyrosine
conjugated
Acceptor Beads
biotinylated
Anti-phage
Streptavidin-coated
Donor Beads
AlphaLISA is as accurate, and more precise,
reliable and less time-consuming than conventional
phage titering. Typical offset between the two methods is
less than half a Log unit.
• AlphaLISA provides an accurate determination of total
phage particles, whereas convential titration is a
biofunctional assay measuring viable phage.
• We demonstrate a close correspondance between these
two values under the phage purification conditions used
here (Sequential PEG precipitation, 0.22µm filtration and
Size-Exclusion Chromatography).
Src wtSrc YF
Src wtSrc YF
Amplified phagefrom culture medium
PEG-purifiedphage
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
10 -6 10 -5 10 -4 10 -3 10 -2 10 -10
Phage dilution
Alp
ha
Sc
ree
n c
ou
nts
(c
ps
)
0
2,500
5,000
7,500
10,000
12,500
15,000
17,500
10 -6 10 -5 10 -4 10 -3 10 -2 10 -10
Phage dilution
Alp
ha
Sc
ree
n c
ou
nts
(c
ps
)
10,000
15,000
20,000Akt wt
Akt SA
103 104 105 106 107 1080
pfu
Alp
ha
LIS
A c
ou
nts
(c
ps
)
Further purification of lytic phage T7 is required for
PhosphoSensor detection
Phage colonies on solid support can be phosphorylated by
kinases, and these experiments represent the first example
for detection of bacteriophage phosphorylation in free solution
•Kinase assay and detection in the same well
•Constructs are specifically phosphorylated
AlphaLISA titering
100 pfu 300 pfu 1000 pfu 1:1 1:100 1:10001:1 1:100 1:1000
Control phage library
Conventional titering
T7 titering samples
Number of phage samples in
one typical experiment
10-3 PHAGE DISPLAY
Bacteriophages can easily and precisely be titrated by
AlphaLISA in significantly less time (4X) than
conventional methods
Total: 1 ½ days
Total: 3 hours
Kinome representation courtesy of
0
50000
100000
150000
200000
250000
Alp
haL
ISA
co
un
ts (
cp
s)
pfu
biotin 0
biotin 1
biotin 2
biotin 3
biotin 4
biotin 5
biotin 6
biotin 7
biotin 8