1
Emission 615 nm biotinylated Anti-phage Streptavidin- coated Donor Beads Anti-M13 conjugated Acceptor Beads 1 O 2 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 LM 3+ LM 3+ LM 3+ LM 3+ Emission 615 nm biotinylated Anti-T7 Streptavidin- coated Donor Beads Anti-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 40 0 100,000 200,000 300,000 400,000 500,000 600,000 700,000 800,000 900,000 1,000,000 1,100,000 1,200,000 Phage samples processed (triplicats) AlphaLISA counts (cps) 0 25,000 50,000 75,000 100,000 125,000 150,000 175,000 10 3 10 4 10 5 10 6 10 7 0 pfu AlphaLISA counts (cps) Standard curve with T7 library biotinylated Anti-T7 Mouse IgG Streptavidin-coated Donor Beads Anti-rabbit IgG conjugated Acceptor Beads Anti-RXRXXpS/pT Rabbit IgG biotinylated Anti-T7 Streptavidin-coated Donor Beads Anti-phospho tyrosine conjugated Acceptor Beads Phage 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 MgCl 2 . 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 Abstract 1 Phage-Expressed Proof-of- Concept Constructs 5 c-Src T7 Phage Substrates 9 2 M13 Phage Titering 6 Akt1 T7 Phage Substrates 10 7 Summary 11 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 Systems 3 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 Methods 4 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 wt KIEEPLYWMFG Src 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 de-orphanization strategy Project Goals M13 T7 0 100 200 300 400 500 600 700 800 900 Src wt Src YF 10 3 10 4 10 5 10 6 10 7 10 8 0 pfu AlphaScreen counts (cps) T7 Phage Titering 8 M13 can easily be titrated by AlphaLISA Many antibody combinations work efficiently 10 8 10 9 10 10 10 11 Phage dilution AMPLIFIED PHAGES PURIFIED PHAGES Conventional 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 wt Src YF Src wt Src YF Amplified phage from culture medium PEG-purified phage 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 -1 0 Phage dilution AlphaScreen counts (cps) 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 -1 0 Phage dilution AlphaScreen counts (cps) 10,000 15,000 20,000 Akt wt Akt SA 10 3 10 4 10 5 10 6 10 7 10 8 0 pfu AlphaLISA counts (cps) 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 1:1000 1:1 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 AlphaLISA counts (cps) pfu biotin 0 biotin 1 biotin 2 biotin 3 biotin 4 biotin 5 biotin 6 biotin 7 biotin 8
