Discovery of Ligands for βγ Subunits from Phage-Displayed Peptide Libraries

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[39] Discovery of Ligands for fly Subunits from Phage-Displayed Peptide Libraries By ALAN V. SMRCKAand JAMIE K. SCOTT Heterotrimeric G protein 13F subunits bind to many different effector proteins.1 The detailed protein-protein interactions involved in/3y binding to these targets are not well understood. One proposed model for interaction between/~ y subunits and effectors suggests that each effector may have shared and unique binding sites on the/3y surface. Evidence in favor of this hypothesis is that activation of all effectors is blocked by GDP bound ot subunits, yet specific site-directed mutants can be found that block activation of some effectors while having little effect on others. 2'3 That unique binding interactions may exist for various effectors suggests that specific ligands could be identified that bind to some of these unique interaction sites and thus specifically inhibit the activation of some effectors by/3F subunits. One method for defining sequences in effectors that bind to 13Y subunits has been to use peptides derived from the effectors in competition-based assays of effector activation. 4,5 Since 13)/ subunits bind to peptides derived from various effectors, we reasoned that combinatorial phage-displayed peptide library screening could identify novel ligands for binding to G protein/3 y subunits at both common and unique effector interaction sites. One such screen in our hands yielded a family of peptides that further defines the protein requirements for interaction at specific sites on/~y subunits. 5a The peptides have a relatively high affinity for/~y subunits and selectively interfere with certain/3y-mediated pathways. The phage bearing the peptides provide a convenient tool for comparing the/~ y binding sites of various effectors to determine if they are shared or unique. Ultimately such ligands could lead to the development of specific small-molecule inhibitors of/~ y subunit mediated pathways. Many phage display methods have been published elsewhere. 6'7 Here we describe many of these methods as they specifically relate J D. E. Clapham and E. J. Neer, Ann. Rev. Pharmacol. Toxicol. 37, 167 (1997). 2 H. E. Harem, J. BioL Chem. 273, 669 (1998). 3 y. Li, P. M. Sternweis, S. Charnecki, T. E Smith, A. G. Gilman, E. J. Neer, and T. Kozasa, J. Biol. Chem. 273, 16265 (1998). 4 j. Chen, M. DeVivo, J. Dingus, A. Harry, J. Li, J. Sui, D. J. Catty, J. L. Blank, J. H. Exton, R. H. Stoffel, J. Inglese, R. J. Lefkowitz, D. E. Logothetis, J. Hildebrandt, and R. Iyengar, Science 268, 1166 (1995). 5 G. Krapivinsky, M. E. Kennedy, J. Nemec, I. Medina, L. Krapivinsky, and D. E. Clapham, J. Biol. Chem. 273, 16946 (1998). 5a j. K. Scott, S. E Huang, B. E Gangadhar, G. M. Samoriski, E Clapp, R. A. Gross, R. Taussig, and A. V. Smrcka, EMBO J. 20, 767 (2001). 6 G. P. Smith and J. K. Scott, Methods Enzymol. 217, 228 (1990).

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to analysis of G protein fly subunits. These methods also have the potential to be directed toward defining ligands for other signal transduction molecules. General Phage and Bacterial Methods

f88-4 Phage Display System The phage display system for these studies uses the phage f88-4, which is a derivative of fd-tet which itself is in the M13 family of filamentous phage. 8 In this system individual peptides or peptide libraries are fused with the N terminus of the pVIII coat protein. Each phage particle contains 2000-2500 copies of the pVIII coat protein. The DNA sequences for the peptides to be displayed are inserted at the 5' end of a second copy of gene VIII in the phage genome under the control of a separate promoter (tac) such that expression of the peptide/pVIII fusion proteins is restricted to 1-10% of the total coat proteins. This multivalent display of roughly 20-200 copies of the peptide per phage particle was crucial to successfully finding ligands that bound to fly subunits. Because any single peptide may have relatively low affinity for/~y, the combined effect yields a particle with a high avidity for the immobilized target. As will be described, if binding of the phage to fly is assessed under conditions that do not allow for multivalent interactions, no binding to /3y subunits is detected (see section on phage ELISA). Compositions of solutions are given in the appendix at the end of this article. F88-4 phage contain an inducible tetracycline-resistant element inserted in the origin of replication for (-)strand synthesis which renders the f88-4 genome replication deficient. As such, cells infected with f88-4 virions are tetracycline resistant and since f88-4 is replication deficient they do not form large plaques after infection of Escherichia coli. For propagation of phage clones, instead of selecting plaques, cells infected with virus are plated on tetracycline and isolated colonies of E. coli infected with phage are selected. Tetracycline resistance under the control of the tet repressor is induced with low levels of tetracycline followed by selection of infected E. coli at a higher tetracycline concentration. Me~o~ Preparation of Starved K91 E. coli Cellsfor Infection with f88-4. Filamentous phage infect E. coli strains displaying the sex pilus encoded by the F episome. The following procedure maximizes display of the F pilus and concentrates the cells for optimal infection. Streak K91 strain of E. coli cells (CGSC#4616; http://cgsc.biology.yale.edu) from a glycerol stock onto NZY agar and incubate overnight at 37 °. Pick an isolated 7 C. E Barbas,J. E. Buss, J. K. Scott,and G. J. Silverman,"PhageDisplay:A LaboratoryManual." Cold SpringHarborLaboratoryPress, ColdSpringHarbor,NY, 2000. 8L. L. C. Bonnycastle,J. S. Mehroke,M. Rashed, X. Gong,and J. K. Scott,J. Mol.Biol. 258, 747 (1996).

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colony from the plate and transfer to a 14 ml snap-cap culture tube containing 2 ml of NZY medium. Incubate the culture overnight at 37 ° in a shaker at 250 rpm. In the morning inoculate 20 ml of NZY medium with 200 #1 of the overnight culture in a 125 ml flask. Shake at 250 rpm at 37 ° until the cells reach OD600 of 0.45 (start to measure the OD after about 1 h and 20 min). Reduce the speed of the shaker to 100 rpm for 10-20 min. Measure the OD600 again, which should be between 0.5 and 0.6. Transfer the culture to a 50 ml sterile conical tube and centrifuge at 600g for 10 min at 4 ° or room temperature. Suspend the cells in 20 ml of sterile 80 mM NaC1 and shake at 100 rpm in a 125 ml culture flask at 37 ° for 45 min to starve the cells. Transfer the mixture to a 50 ml sterile conical tube and centrifuge at 850g for 10 min at 4 °. Gently suspend the cells in 1 ml of 4 ° NAP buffer. Keep the cells on ice until ready for use. The cells can be stored on ice for up to 1 week but are best if used when freshly prepared. Propagation and Small-Scale Preparation of Phage Particles. Dilute the desired phage (clones or pools) to 1 x 102-1 x 104 phage particles/#l in TBS-gelatin and mix with 10/zl of starved cells in an 1.5 ml microfuge tube and incubate at room temperature for 15 min (always use aerosol resistant tips when pipetting phage). Add 500/zl NZY medium containing 0.2/zg/ml tetracycline and incubate at 37 ° for 30 min in a shaker with the tube inverted in a beaker. Add 250/zl of NZY medium containing 15/zg/ml tetracycline and spread 100/zl on NZY agar containing 40/zg/ml tetracycline and incubate overnight at 37 °. To amplify phage for a binding experiment or sequencing, isolated colonies are selected from the NZY-tetracycline plates and inoculated into 1.5 ml of NZY medium containing 15 /zg/ml tetracycline in a 14 ml culture tube and grown overnight at 37 ° at 250 rpm. After removal ofE. coli cells by centrifugation, 1.2 ml of the supernatant is transferred to a fresh microfuge tube and the phage particles are partially purified by addition of 180 #1 of polyethylene glycol (PEG)/NaC1 followed by vortexing and incubation for 2 h on ice. Samples are centrifuged for 40 min at 12,000 rpm in microfuge at 4 °. The supernatant is aspirated and the pellet is suspended in 100 #1 of TBS. The tubes are heated to 70 ° for 30 min to kill residual E. coli cells and centrifuged for 10 rain at 12,000 rpm at 4 °, and the supernatant is removed to a new tube. The final phage concentration from this procedure is generally around 1 x 101° phage particles//zl. Phage particles should be stored at 4 ° for several months or can be stored for a longer term at - 2 0 ° if glycerol is added to 50%. For confirmation of the sequence of the phage at the same time as the phage particles are prepared, 3 ml cultures are grown and 1.5 ml of the culture supernatant is used to prepare ssDNA for sequencing using Qiagen MI3 single-stranded (ss) DNA purification kit, and 1.2 ml is used for phage particle preparation. Phage concentrations can be estimated by agarose gel electrophoresis of the purified phage and ethidium bromide staining of the phage DNA. A standard curve is generated with f88-4 phage that had been grown in large scale, purified twice by PEG precipitation and quantitated spectrophotometrically 6 (100 ng

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DNA --~ 1 × 101° phage particles). Two/zl of the phage preparation is diluted to 8/zl with TBS followed by addition of 2 #l of 5× phage lysis mix. The sample is heated to 70 ° for 20 min followed by electrophoresis of the entire sample on a 1% agarose gel containing 4× GBB and 0.5 #g/ml ethidium bromide with f88-4 phage standard corresponding to a range of 5 × 109-4 × l01° phage particles loaded on the gel. The gel is run for 4 h at 20 V. Phage concentrations can also be estimated by titering the phage and given that the ratio of total phage particles to infectious phage particles is about 20 : 1. Starved cells are infected with phage serially diluted in TBS gelatin as described above. Rather than spreading 100 kel of the mixture on the plate, spot 15/zl of 4 - 5 dilutions on separate areas of a 100 mm dish with NZY-40 lzg/ml tetracycline in agar. If the plates are put at 37 ° overnight the colonies often become too large and merge with each other, making it impossible to count the colonies. To overcome this, the plates are incubated overnight at room temperature in the dark. In the morning the plates are placed in a 37 ° incubator and the growth is monitored throughout the day until the colonies reach a size that is easily discernable. P r e p a r a t i o n o f fly S u b u n i t s for S c r e e n i n g o r E L I S A Prior to screening or phage enzyme-linked immunosorbent assay (ELISA) the fl Y subunits are immobilized in the wells of a 96-well microtiter dish. For screening we have used fly subunits immobilized via covalently attached biotin. For the ELISA we have successfully used both immobilization via biotin or immobilization of the fly directly to the plastic well of the dish.

fl y Biotinylation Sf9 Culture and Membrane Preparation. Biotinylated fllY2 subunits are prepared using a modification of the method developed by Dingus et al. 9 Sf9 (Spodopterafrugiperda ovary) insect cells grown in suspension are infected with His6-oql, fib and )/2 with modifications to what has been previously described by Kozasa and Gilman. l° The sf9 cells are grown in 800 ml Sf 900 II medium (Gibco-BRL, Gaithersburg, MD) in a 2 liter culture flask with shaking at 125 rpm at 27-28 °. The cells are infected at a density of 2-3 × 106 cells/ml with 7.5 ml of His6, otil, 5 ml fib and 2.5 ml Y2 viruses at approximately 1 × 108 pfu (plagueforming units)/ml each. The insect cells from the 800 ml culture are harvested by centrifugation in 500 ml culture bottles in a Beckman JA l0 rotor at 2400 rpm. The cells are suspended in PBS (10 m M KPO4, pH 7.4, 150 mM NaC1) and transferred to a 50 ml conical culture tube and centrifuged at 3000g for 30 min at 4 °. The 9 j. Dingus, M. D. Wilcox,R. Kohnken,and J. D. Hildebrandt,Methods Enzymol. 237, 457 (1994). l0 T, Kozasaand A. G. Gilman,J. Biol. Chem. 270, 1734 (1995).

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supernatant is discarded and the cell pellets either frozen in liquid N2 and stored at - 7 0 ° for later use, or suspended for immediate processing in 15 ml of 4 ° lysis buffer [50 mM HEPES, pH 8.0, 0.1 mM EDTA, 100 mM NaC1, 10 mM 2-mercaptoethanol, 10/zM GDP, and 100/zg/ml phenylmetbylsulfonyl fluoride (PMSF)]. If the cells are removed from storage at - 7 0 ° they should be suspended in room temperature lysis buffer to facilitate thawing of the cell pellet. The suspension is frozen and thawed 4 x by alternately plunging the 50 ml tube containing the suspension into liquid N2 and thawing in a 37 ° water bath. The lysate is then diluted to 100 ml with lysis buffer and the particulate fraction containing membranes is harvested by centrifugation at 100,000g for 45 rain at 4 °. The supernatant is discarded and the membranes are suspended in 60 ml of extraction buffer, 50 mM HEPES, pH 8.0, 3 mM MgCI2, 50 mM NaC1, 10 mM 2-mercaptoethanol, 10/zM GDP and 100/xg/ml PMSF, using a Dounce homogenizer. Extraction and Partial Purification of G Protein Heterotrimer. Proteins are extracted from the membrane fraction by addition of cholate to 1% to the suspended membrane fraction with slow stirring at 4 ° for I h. Detergent-insoluble particulate matter is removed by centrifugation at 100,000g for 45 min at 4 °. The supernatant is diluted 5-fold with 20 mM HEPES, pH 8.0, 100 mM NaC1, 0.5% polyoxyethylene 10 lauryl ether (C12EI0), 1 mM MgCI2, 10 mM 2-mercaptoethanol, 10 # M GDR 100 #g/ml PMSF, and loaded onto a 2 ml Ni-NTA agarose column at 0.5 ml/min overnight. The column is washed with 80 ml of 20 mM HEPES, pH 8.0, 1 mM MgCI2, 10 mM 2-mercaptoethanol, 10/zM GDP, 300 mM NaC1, 5 mM imidazole, 0.5% C12EI0, and 100 #g/ml PMSE Heterotrimeric His6-otlfl~/2 is eluted from the column with six successive 2 ml aliquots of 20 mM HEPES, pH 8.0, 100 mM NaC1, 0.1% C12El0, 10 # M GDP, and 150 mM imidazole. The eluted fractions are assayed for protein using an amido black protein assay u and the fractions are separated on a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel and stained with Coomassie blue. Fractions containing the largest amount of fly are used for the biotinylation reaction.

Biotinylation of OliflY 2 Heterotrimer and Purification of Biotinylated f y Subunits. The His6-otiflY2 eluted from the Ni-NTA-agarose is diluted to 1 mg total protein/ml with 20 mM HEPES, pH 8.0, 1 mM EDTA, 1 mM DTT, 100 mM NaC1, 10/zM GDP, and the final detergent concentration is adjusted to 0.05% C12E10. NHS-LC-biotin (Pierce, Rockford, II) is added from a 20 mM stock in dimethyl sulfoxide (DMSO) to give a final concentration of 1 mM. The reaction is allowed to proceed for 30 min at room temperature followed by addition of 10 mM ethanolamine pH 8.0 from a 200 mM stock and incubation on ice for 10 min. The sample is diluted to 90 ml with dilution buffer: 20 mM HEPES, pH 8.0, 100 mM NaC1, 10/zM GDP, and 0.5% C12E10. Two ml of washed Ni-NTA agarose is added and incubated with mixing overnight at 4 °. The mixture is poured through a column 11W. Schaffner and C. Weissmann, Anal. Biochem. 56, 502 (1973).

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and washed with 20 ml of dilution buffer. The column is warmed to room temperature for 15 min and washed with 5 ml of 30 ° wash buffer, 20 mM HEPES, pH 8.0, 100 mM NaC1, 10 # M GDP, and 1% cholate, flF subunits are eluted with 5 successive 2ml aliquots of 30 ° wash buffer plus 10 mM MgC12, 10 mM NaF, and 30/zM A1C13. The eluted fly is detected by electrophoresis of the fractions on a 12% SDS-polyacrylamide gel followed by staining with Coomassie blue. Biotinylation is confirmed by electrophoresis and blotting with streptavidin-horseradial peroxidase (HRP) and by showing that all of the biotinylated fly could be bound to streptavidin agarose. For screening of libraries and ELISA assays the b-fly is stored in the elution buffer. To test the viability of the fly in other assays the biotinylated fly may have to be exchanged into another buffer. Libraries Procedures for construction of phage displayed pepfide libraries in f88-4 have been described. For detailed procedures for preparation of the libraries see (Ref. 7). Some libraries are available from Dr. George Smith's laboratory including an f88-4 linear 15-mer library and several types of cysteine constrained libraries as well as wild-type f88-4 virus (see Smith laboratory Web site for details (http://www.biosci.missouri.edu/smithgp/)]. Other phage display libraries are available commercially but use plII-based peptide display (New England Biolabs, Beverly, MA).

Amplification of Libraries Inoculate a 125 ml culture flask containing 15 ml NZY with a single fresh colony of K91 cells and shake overnight (250 rpm) at 37 °. In a 2 liter culture flask, inoculate 300 ml NZY with 6 ml of the overnight K91 culture. Shake at 250 rpm until the cell concentration reaches OD600 of 0.45. Slow the shaker to 100 rpm for 10 min to allow the cells to regenerate their pili. Measure the OD60o; it should not be over 0.65 (aim for 0.55-0.65). At OD60oof 0.6, the cell concentration equals --~4 x 108 cells/ml, yielding at total of -~ 1.2 x 1011 cells. Transfer the cells to two 250 ml centrifuge bottles, and centrifuge them at 1000g for 10 rain. Pour off the supernatant, briefly centrifuge the bottles again, and remove the remaining supernatant. Gently suspend each pellet with 150 ml 80 mM NaC1 and transfer to a sterile 500 ml culture flask. Incubate the mixture for 60 min at 37 ° with shaking at low speed (50 rpm). Transfer the cells to two 250 ml centrifuge bottles, and spin them at 1100g for 10 min at 4 °. Resuspend each pellet in 5 ml ice-cold NAP buffer, then transfer the cells to a 14 ml snap-cap tube. Wash each bottle out in sequence with a single aliquot of 1 ml of NAP buffer and transfer this to the snap-cap tube. This should give a final concentration of 10 l° cells/ml in 11 ml. Store on ice at 4 °. Remove 30/zl of the starved cells to a 1.5 ml microfuge tube containing 10 6 f88-4 control phage particles to serve as a positive control infection. Add 2 x 10 l°

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phage particles from the library to be amplified to the remaining cells and mix by gentle inversion [this should give a multiplicity of infection (MOI) of ~5 cells per phage particle]. Let the infections stand for 10 min at room temperature with occasional gentle swirling. Briefly centrifuge at 1000g for 1 min at 4 ° on a tabletop centrifuge to bring down the droplets on the walls of the tube. Mix the cells with a 1 ml pipette. Add 0.5 ml of infected cells to each of 22 125-ml flasks with each containing 45 ml NZY and 0.2/zg/ml tetracycline. Shake the cultures at 250 rpm at 37 ° for 30-45 min to induce tetracycline resistance. Add 333 #1 of 2 mg/ml tetracycline to each culture to bring the tetracycline concentration to 15/zg/ml. Mix and remove 20 #1 samples from a few cultures for titering. This will quantitate the number of infected cells that will produce the library and hence the number of phage clones comprising the amplified library. Shake the cultures at 250 rpm overnight (20 h) at 37 °. Combine the library cultures in four 250 ml centrifuge bottles. Centrifuge at 2400g for 10 min at 4 °. Pour off the supernatant into clean bottles, being careful not to disturb the cell pellet, and recentrifuge at 6000g for 10 min at 4 °. Carefully pour the supernatant into tared, 250-ml centrifuge bottles and note the culture volume in each bottle (1 g = 1 ml). To each bottle, add 0.15 volume polyethylene glycol (PEG)/NaC1. Screw on caps tightly, and mix throughly by inverting the bottles gently ~ 100 times. Incubate the mixtures on ice for >4 h on ice or overnight at 4 °. Pellet the phage by centrifuging at 6000g for 40 min at 4 °. Pour off and discard the supernatant, being careful not to disturb the pellet. Remove the residual supernatant by briefly recentrifuging the bottles, tilting each bottle so that the pellet is opposite the remaining supernatant, and aspirating the residual supernatant with a P1000 Pipetman. Add 7.5 ml TBS to each bottle and shake at 150 rpm in a 37 ° incubator for ~30 min to dissolve the pellets. Centrifuge the bottles briefly to drive the solution to the bottom of each bottle. Transfer the solution from two bottles to a single tube, yielding two tubes. Rinse the bottles with another 7.5 ml TBS and add to each tube, as before. Balance the tubes with TBS and mix the phage thoroughly by inversion. Centrifuge the tubes at 10,000-20,000g for 10 min at 4 ° to clear the supernatants. Transfer the supernatants to fresh, tared tubes; note the volumes. Add 0.15 volume PEG/NaC1 to each tube, and invert gently ~100 times. Allow the phage to precipitate by incubating the tubes on ice for > 1 h (a heavy precipitate should be evident at the end of that time). Collect the precipitated phage by centrifuging at 10,000g for 40 min at 4 °. Carefully and completely remove the supernatant. Add 10 ml TBS to each tube, and dissolve the phage pellet by gently vortexing, then allowing the pellet to soften at room temperature for --~1 h. Vortex again, and briefly centrifuge to drive the solution down. If the phage are to be further purified on a CsC1 density gradient, add only 5 ml TBS to each tube and resuspend the phage as above. Combine the two supernatants into a single round-bottom polypropylene centrifuge tube. At this point, the phage can be heat treated to kill any remaining cells. Incubate the tubes for 30 min in a 70 ° water bath. (Note: The heat-treatment step is optional and may

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denature complex proteins displayed by some phage.) This step is not necessary if the phage are to be CsC1 purified. Clear the supernatants by centrifuging the tubes at 10,000-20,000g for 10 min at room temperature or 4 °. Pour the cleared supernatant from each tube into a 15-ml polypropylene snap-cap tube and store at 4 ° in the dark. To determine the concentration and the yield of phage particles, treat an aliquot of phage with 5 x Lysis mix, then dilute the mixture in 1 × Lysis mix and run the samples on a 1.2% agarose gel in 4× GBB, using a known amount of control f88-4 phage treated in the same way as a standard. The concentration of phage particles can be more accurately assessed by spectrophotometric analysis; however, this is better done with CsCl-purified phage. The final concentration of phage should not exceed ~3 × 1013/ml, so once the phage concentration is known, it should be adjusted accordingly with TBS. To impede cell growth, the solution can be adjusted to a final concentration of 0.02% (w/v) NaN3 (sodium azide, a highly toxic poison), or 20 mM Na2EDTA. The phage can be stored long term in 50% (v/v) sterile glycerol at - 1 8 °. Screening of Phage-Displayed Peptide Libraries against G Protein/~), Subunits Biotinylated fly subunits are immobilized in the wells of a microtiter plate coated with streptavidin. Prior to screening, the libraries are preadsorbed to wells of a plate containing l /zg of streptavidin to reduce the possibility of obtaining streptavidin binders from the screen. To determine the degree to which specific fly binding clones are enriched with each stage of panning, the libraries are also screened in wells that do not have immobilized fly and equal numbers of f88-4 wild-type phage are screened in/~y subunit-containing wells in parallel with the actual library screen against immobilized/3 y.

Preabsorption of Libraries with Streptavidin For each screening condition one well of a 96-well microtiter plate (Coming/ Costar, Coming, NY) is coated with streptavidin (Sigma, St. Louis, MO) by incubating the plate overnight at 4 ° with 35 #1 of TBS containing 1/zg of streptavidin. The streptavidin solution is removed and 200 #1 of 2% dialyzed bovine serum albumin (BSA) in TBS is added to each well and incubated for 30 min at 4 °. The BSA solution is then removed and the plate is washed 3 times with TBS. For each wash throughout this procedure a wash bottle is used to completely fill the wells being careful not to cross contaminate the wells, and the wells are emptied by shaking into a sink followed by slapping the plate face down on a paper towel to remove any remaining wash solution. Immediately after slapping the last wash from the plate add 103-104 equivalents of each phage library and 2% B SA in TB S/1.5 % Tween 20 to bring the final volume/well to greater than 35/zl/well and the final concentration

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of Tween 20 to 0.5%. For example, if the library has a diversity of 1 x 108 clones, 1011-1012 phage particles should be added to each well. Add the BSA/TBS/Tween to each well, then add the library using filtered pipette tips to transfer the library samples from their stock solutions. Incubate the plate 3-4 h at room temperature with rocking. Transfer the preadsorbed phage solutions to microfuge tubes.

Screening Libraries A second plate that has been coated with streptavidin as described above is used for screening the library. The streptavidin solution is removed by aspiration and the wells are washed once with TBS. To each well is added 200/zl of 2% BSA in TBS followed by incubation at 37 ° for 1 h. The wells are washed three times with TBS/0.5% Tween 20 and 35/zl of biotinylated 50 nM/3y subunit is added in TBS/0.5% Tween 20 for 40 min at 4 °. After the incubation with b-/3y, 10 #1 of 1 mM biotin in TBS/Tween 20 containing 2% BSA is added to each well and incubated for 10 min at 4 ° to block unbound biotin binding sites on streptavidin. Remove the/3y solution and wash the plate three times with TBS/Tween 20. Transfer the preadsorbed libraries to the wells containing fly subunits and incubate 2 h at room temperature. After the 2 h incubation aspirate the phage from the wells with a pipettor and wash the wells six times with PBS/Tween 20 with a 2 min incubation for each wash. After the last wash add 35 /zl phage elution buffer (0.1 M HC1 adjusted to pH 2.2 with glycine, 1 mg/ml BSA) to each well and mix with a pipette. Incubate 10 min at room temperature. Remove the eluate, transfer to another 96-well plate, and neutralize with 5-7 #1 of 1 M Tris, pH 9.1. Verify that the pH has been neutralized to pH 7-8.5. To amplify the eluted phage and to assess the yield of phage from the screen, K91 E. coli cells are infected with the eluted phage and known amounts of the control phage. Add 10/~1 of starved K91 cells (made that same day) to the eluted phage from the control samples, the screening samples, and 106 particles of f88-4 phage vector and allow the infection to go for 15 min. The control samples are used solely for titering purposes and calculation of phage yields and they are not amplified for further rounds of screening. To induce tetracycline resistance add 140 #1 of LB/0.26/zg/#l tetracycline to each infection reaction. Cover the plate with a lid and place in a humidified plastic box and shake for 30 min at 37 °. After the tetracycline resistance genes are induced add 20/zl of LB containing 148/zg/ml tetracycline. At this point the yield of phage from the selection is determined by titering 5/zl from each infection. After the first round of panning the phage yield should be about 10-4-10-5% (104 or 105 phage particles if l0 II phage particles are used for the screening). The titering is done in a range of 1 : 10 to 1 : 10,000 dilutions of phage in LB according to the procedure described in the section on propagation and preparation of phage particles. The remainder of the cells are incubated at 37 ° with shaking for 42 h to amplify the eluted phage. To calculate the percent phage yield,

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the efficiency of the infection is determined from the control infection with 106 particles of f88-4 (infective units derived from the titer/106 input phage particles). The infection efficiency is then used to determine the actual input tranducing units (TU) for each library (input TU equals the phage particles added to each well for the screening times the infection efficiency). The percent yield of the panning step is calculated by dividing the output TU determined from titering the eluted phage by the input TU (calculated above). The degree of enrichment is calculated by comparing the phage yields from the control panning (no/~y in the well or fly screened with f88-4) with the phage yield from the actual screening well. At early stages of panning there may be very little enrichment of phage relative to control samples without biotinylated fly, but with subsequent panning steps the yield of phage should be enriched relative to a control panning. This gives an idea of whether the panning steps have been successful. After the 42-h amplification period the samples of amplified phage samples are harvested, the E. coli cells removed by centrifugation, and the supernatant is treated at 70 ° for 20 min to kill residual bacteria. The sample is centrifuged again to remove dead cells. Twenty #1 of each culture supernatant is mixed with 90 #1 of 60% glycerol and stored at-20 °. Twenty #1 of the supernatant is used for gel analysis with known amounts of control phage as described in the section on propagation and small-scale preparation of phage particles. This will help determine how much of the phage to use for the next round of panning. Rounds 2, 3, and 4. Follow the same procedures as for round 1 with following exceptions: 1. Sincethe number ofclonesin the pools ofenriched phage is relatively small compared to the number going into the first round of panning, fewer phage can be used in the subsequent rounds of screening. Use 10- to 100-fold fewer input phage than was used in the round 1 screening while keeping the volume at 35/zl and the concentration of Tween 20 in the preabsorptions as close to 0.5% as possible. 2. For each screen use the same amount of F-88 control phage as is used for the panning of the amplified phage pools. The phage from the control pannings in round 1 are not supposed to be used for anything but titering and determining the phage enrichment. Do not use these for subsequent rounds of panning. 3. Consider performing ELISA on eluted amplified phage pools to determine if there are increases in binding compared to vector-control phage. The highest sequence diversity and, possibly, the best binders can be present in rounds that precede rounds having yields that are above background. After the third round of screening the amplified pools of selected phage should bind to/~y subunits. The pools should be checked by performing an ELISA in the

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presence and absence of immobilized/3y. The binding should be compared with the binding of wild-type f88-4. If significant binding is observed above the f88-4 and -fly controls, the pools are diluted and used to infect K91 cells and spread on NZY tetracycline plates to give well-separated colonies as described in the section on propagation and small-scale preparation of phage particles. Individual colonies representing individual phage clones with unique peptide sequences are picked and grown overnight in 3 ml of media. The culture (1.5 ml) is used for phage preparation and purification by PEG precipitation as described. These clones of phage are then tested for binding for 132/in the ELISA assay with the appropriate controls. If significant binding is observed, single-stranded DNA is prepared from the remaining 1.5 ml of culture supernatant using a Qiagen M13 single-stranded DNA preparation kit (or other method) for DNA sequencing. Results from Screening. The phage selected from the libraries that bound to fly subunits had multiple sequences with various levels of homology to one another. For our analysis we screened several different types of libraries of various lengths and that had differing internal disulfide constraints, s Some of these sequences grouped into families are shown in Fig. 1. We have shown that synthetic peptides constructed on the basis some of these sequences are able to inhibit/~y subunitmediated regulation of phospholipase C-/~ (PLC-/~) and phosphatidyl inositol 3-kinase y but not adenylate cyclase I or a Ca 2+ channel. 5a Our model is that these peptides are binding at a unique site utilized by some effectors but not others. C o n s t r u c t i o n of P h a g e D i s p l a y i n g S e l e c t e d P e p t i d e s Phage bearing specific peptide sequences can be constructed by inserting complementary oligonucleotides encoding the desired sequence in frame with the gene encoding the second copy of the pVIII coat protein. Also encoded in the oligonucleotide is a portion of the leader sequence and PstI and HindlII cloning sites. Leader cleavage site M L S F A N V P A E G D D . . . . . . . ATG CTA AGC TTT GCC AAC GTC CCT GCA GAA GGT GAT GAC HindlII PstI 5'-AGCTTTGCC XXXXXXXXXGCTGCA-3' AACGG XXXXXXXXXCG The export signal sequence LSFA has to remain intact, NV are lost, and the phage sequence resumes with AAE. For cloning of the insert, the double-stranded replicative form of the f88-4 viral DNA must be purified by standard methods 6 and cleaved with HindlII and PstI.

568

G PROTEIN STRUCTUREAND FUNCTIONALDOMAINS

I

[39]

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FIG. 1. Alignmentsof sequences obtained by random peptide phage display screening. Sequences were placed into four groups based on sequence similarity. Black boxes indicate identities and gray boxes indicate conservativesubstitutions. Reproducedfrom J. K. Scott et al., EMBO J. 20, 767 (2001) with permission of Oxford UniversityPress.

The oligonucleotides are annealed and then ligated into the cleaved f88-4 vector DNA. The ligated DNA is transformed into MC1061 E s c h e r i c h i a c o l i by electroporation or other methods. After electroporation, tetracycline resistance is induced by addition of 1 ml of NZY with low tetracycline (0.2 mg/ml) and shaking for 1 h at 37 °. Two hundred /zl of cells is then plated on NZY agar containing 4 0 / z g / m l tetracycline and incubated overnight at 37 °. Isolated colonies are picked and grown overnight in 3 ml NZY medium with 15/xg/ml tetracycline. Of the overnight culture, 1.5 ml is used for preparation of single-stranded phage

[39]

LIGANDS FOR fly SUBUNITSFROM PEPTIDELIBRARIES

569

DNA (Qiagen M13 DNA preparation kit) followed by sequencing to confirm in frame fusion of the insert. The sequencing primer we have used has the sequence 5'-CTGAGTFCATTAAGACG-Y. The remainder of the culture is saved and used to prepare phage particles as described in the section on propagation and smallscale preparation of phage particles. Based on our estimates of the affinity of the selected peptides for fly subunits we would predict that most peptides with an affinity of 100 # M or greater would bind in this assay. On the other hand we have fused peptide sequences from K + channels described by Krapivinsky et al. 5 but have been unable to detect binding. We suspect that there are steric constraints placed on the peptide by fusion to the pVIII coat protein that prevents interaction with fly. To overcome this problem we are inserting spacer sequences between the coat protein and the displayed peptide so that the peptides will be more available on the surface of the phage for interaction with the fly subunits. A n a l y s i s o f B i n d i n g to fly S u b u n i t s u s i n g P h a g e ELISA To examine phage binding to fly, the fly subunits are immobilized in the wells of a microtiter plate. Selected phage particles representing single clones or pools of clones are then allowed to bind to the immobilized fly followed by washing and detection of the bound phage with an antiphage antibody. The fly subunits either can be immobilized directly or can be bound to immobilized streptavidin via covalently attached biotin. The biotinylation approach ensures that the fly is in an active conformation when it is immobilized, increasing the chances that sites required for binding are available to the peptide. Binding of the fly subunits directly to the plate could lead to partial denaturation of the protein and exposure of nonspecific sites, but the procedure is simpler because the reactions involved in biotinylation of fly do not have to be performed. Another ELISA method that has been used to detect binding for phage to target molecules is to immobilize the phage in the bottom of the well and detect binding of soluble target with an antibody directed against the target molecule. This approach measures the ability of the target molecule to bind monovalently to the immobilized phage since the analyte in solution can only bind to one immobilized phage particle at a time. When the target is immobilized, since the f88-4 phage has multiple displayed peptides it can bind to multiple immobilized target molecules simultaneously, resulting in increased avidity. We have been unable to detect binding of 13Y to the phage that we have selected when the phage was immobilized, indicating that the use of a polyvalent phage display system is essential for detecting phage binding to fly subunits with any of the peptides we have identified. ELISA Assay

One/zg of streptavidin is added to each well to be tested in a 96-well microtiter dish in 40/zl of TBS. Control wells are set up that either do not contain/3y subunits

570

[39]

G PROTEIN STRUCTURE AND FUNCTIONAL DOMAINS

or will be screened with wild-type f88-4 phage with no peptide displayed. These wells should also be coated with streptavidin. Each condition is tested in duplicate. After overnight incubation with streptavidin at 4 ° the streptavidin solution is aspirated and replaced with 35/zl of a solution of TBS containing 0.1-0.5% Tween 20 and 2% BSA for 1 h at 4 °. After 1 h the BSA solution is aspirated and the wells are washed 3 times at with TBS 0.1-0.5% Tween 20. For each wash a wash bottle is used to fill the wells and the wells are emptied by shaking into the sink followed by slapping the plate face down on a paper towel to remove any remaining wash solution. After washing, 50-100 ng of b-fly in 35/zl of TBS-Tween 20 is added to the streptavidin-coated wells and incubated for 1 h at 4 °. After incubation, the f y solution is aspirated, the wells washed three times with TBS/0.1-0.5% Tween 20 and 1 × 101° phage are incubated with the immobilized fly in TBS/0.1-0.5% Tween 20 for 1-4 h at 4 °. Overall, the ELISA assays for phage binding are relatively insensitive to phage number but it is still a good idea to use a standard known amount of phage for each binding assay (see Fig. 2). For each incubation 35/zl of TBS-Tween 20 is added to the well followed by direct addition of phage in 1-2/zl. For competition ELISAs, peptides or other competitors are added to the wells just

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log phage number/well (x 10 -10) FIG. 2. Binding of phage displaying peptides to fly subunits at various concentrations of phage particles. Fifty nM biotinylated # y was bound to streptavidin-coated 96-well plate and various amounts of phage clones displaying two different peptide sequences were analyzed in the phage ELISA assay as is described in the text. Black bars are wild-type f88-4 phage controls; gray bars are two different clones displaying fly binding peptides.

[39]

LIGANDS FOR/3Y SUBUNITSFROM PEPTIDE LmRARIES

571

prior to addition of the phage and the incubations only go for 1 h. After washing three times to remove unbound phage, anti-M13 antibody (Amersham Pharmacia Biotech, Piscataway, NJ) linked to horseradish peroxidase is added to each well in TBS/0.1-0.5% Tween 20 with 2% BSA (1:5000 dilution) and incubated at 4 ° for I h. The wells are washed three times with TBS-Tween 20, 40/zl of ABTS is added, and the color reaction allowed to proceed for 5-30 min. The extent of the color reaction is monitored in a microplate reader at 405 nm. With nonbiotinylated/3F subunits 0.05-1 # g of/3y is immobilized in the well directly in TBS instead of streptavidin. The/3F must diluted at least 10-fold into TBS prior to addition to the well since detergent in the/3F solution will inhibit binding to the well. All other steps in the procedure are the same. A comparison of the results of these two methods is shown in Fig. 3. Competition ELISA. One of the powerful uses of the displayed/3F binding peptides on phage is the ability to test whether other molecules to compete for the binding of the phage to 13F subunits. For example, peptides derived from other effectors could be tested for their ability to block the binding of a particular phage clone. If the peptide does compete for binding of the phage then it must be binding to a site that overlaps with the binding site for the peptide that is displayed on the phage. If the peptide does not block the interaction but is known to bind to 13F subunits then it must be binding at a different site on the/3 Y surface. If, for example, the phage clone used for the analysis displays a sequence known to bind to a PLC-/3 interaction site, any peptides that compete for binding of this phage must be binding to a site that is shared by PLC-/3. If the peptide does not compete for phage binding but does compete for/3F-mediated PLC activation, then it binds at site shared by PLC-/3 but distinct from the epitope shared by the phage-borne sequence. In theory any peptide from any G protein/3Y subunit coupled effector could be fused to this phage and competition analysis could be used to determine which effector peptides share this particular interaction site. The procedure for the competition ELISA is identical to what has been described for the phage ELISA above, except peptides or other competitors are added just prior to addition of the phage and the incubations with phage are only 1 h. A n a l y s i s of I s o l a t e d P e p t i d e s in F u n c t i o n a l A s s a y s The phage ELISA assay is a convenient assay for monitoring the ability of a phage-displayed peptide to bind to/3F subunits in a semi high-throughput format, but the method has some disadvantages. One major disadvantage is that the assay does not give any quantitative information about the affinity of the peptide for the target molecule. Additionally, it is important to test the binding of the displayed peptide outside the context of the phage coat protein to be assured that the displayed sequence is all that is required for binding. There are a number of biophysical methods for measuring synthetic peptide interactions with proteins, including

572

G PROTEIN STRUCTURE AND FUNCTIONAL DOMAINS

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fluorescence polarization and surface plasmon resonance. Another approach that we have taken is to test the isolated synthetic peptides in competition-based effector assays where the peptide may compete with the activation of an effector by fly subunits. One system that we routinely use for this analysis is based on the ability of fly subunits to activate PLC-fl isoforms in vitro. Other assays where

[39]

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[peptide] pM FIG. 4. Phage displayed peptides inhibit activation of PLC-/~2fly subunits. A synthetic peptide with the sequence SIRKALNILGYPDYD was included in phospholipase C assays at the indicated concentrations. Reactions included either no ~ 1Y2 (A) or 100 nM~ 1~/2(A) and l ng of PLC-fl2; 100 nM free Ca 2+ and were for 5 rain. Each data point is the mean of duplicate determinations. Reproduced from J. K. Scott et al., EMBO £ 20, 767 (200t) with permission of Oxford University Press.

competition with fly can be tested include assays of PI 3-kinase y activation and adenylate cyclase regulation. Here we describe the assay to test if peptides derived from phage display can inhibit the ability of/~y subunits to activate PLC-/~2. We test the peptides over a range of concentrations in the presence and absence of a fixed concentration of fly. The assay in the absence o f / ~ , is to test whether the peptide has any direct effects on the PLC activity or substrate vesicles. An example of a concentration curve for inhibition of PLC activation by/~ y subunits by a phage display derived peptide is shown in Fig. 4.

Phospholipase C Peptide Competition Assay The total reaction volume for each assay is 60/zl. Each of the following components is added separately to the final reaction in this order: 1. 20 /zl Lipid vesicles containing both unlabeled phosphatidylinositol 4,5bisphosphate (PIP2) and [3H]PIP2 as well as phosphatidylethanolamine (PE). This assay is based on the ability of PLC to release [3H]IP3 into the aqueous phase. 2. 10/zl of solution containing PLC /7 as well as other necessary reaction components. 3. 10/zl of peptide solution at the desired concentration. 4. 10/zl/~y subunit containing solution. 5. 10/zl Ca 2+ solution (calcium is required for the enzymatic activity of the PLC).

574

G PROTEINSTRUCTUREAND FUNCTIONALDOMAINS

[39]

Lipid Vesicles. PIP2 (Avanti) at 50 /xM and PE (Avanti, Alabaster, AL) at 200/zM are the final target concentrations for these lipids in each reaction. Vesicles are made at 150/zM PIP2 and 600/zM PE because 20/zl will be diluted into a final volume of 60/xl. We also need to add [inositol-2-3H(N)]PIP2 ([3H]PIP2) (Dupont NEN, Boston, MA) such that each assay has 4000-8000 cpm. A general calculation to give the volume of stock [3H]PIP2 to be added is to estimate the total volume of lipid vesicle solution needed for the experiment and multiply by 0.03 to give the volume of stock [3H]PIP2 to be added. For example, if we make 450/zl of lipids we would multiply 450 x 0.03 to get 13.5 #1 of 3H-labeled PIP2 to be added. Note: Unlabeled stock lipids are stored in glass vials (Kimble, Vineland, NJ) with chloroform-resistant screw caps (Kimble). We have noticed loss of [3H]PIP2 cpm if it is stored in these vials so we aliquot the [3H]PIP2 into 0.5 ml Nensure vials obtained from New England Nuclear. All the lipids are stored in desiccators at - 2 0 °. The lipids in chloroform are measured and transferred from the stock vials to a glass Pyrex tube with a Hamilton syringe. The lipids are dried under a stream of N2 gas and sonication buffer is added at the final volume that has been calculated. Sonicate 5 min in a sonicating water bath. We find that if 20% Alconox liquid detergent is added to the water bath the sonication efficiency is greatly increased. The final lipid vesicles should be a translucent homogenous suspension without any visible particulate matter and all the lipid suspended from the bottom of the tube. Ten/zl of the vesicle suspension is analyzed by liquid scintillation counting to verify that each assay will receive the proper number of counts per minute of [3H]PIP2. PLCMixture. The phospholipase C in this assay will be used at a concentration of 1-5 ng (depends on the assay) per sample. We usually use PLC-f2 but PLC-f3 can also be used. Each sample will get 10/zl of PLC solution. Make up enough PLC solution for all the reactions by multiplying the total number of reactions by 10 and adding some extra. Mix one-half volume of 2x assay buffer, 2 mM dithiothreitol (DTT), 6 mg/ml BSA, and 0.6% octyl-fl-D-glucopyranoside (OG), the required amount of PLC, and sufficient water to achieve the final desired volume. f), Solution. The stock f ) , is in stored in a buffer that contains 1% OG. We usually exchange the f y from the buffer used for purification from Sf9 cells into the f ) , storage buffer by binding the f ) , to a small (500/zl) column of hydroxyapatite (BioRad), washing with 5 ml of 20 mMHEPES, pH 8.0, 1 mM DTT, 100 mMNaC1, and 1% OG and during with the same buffer containing 200 mM KPI, pH 8.0. Ten/zl of incubation solution containing Ely is added to each reaction tube and therefore we need to make the concentrations of fly 6 x the final concentration that is desired. The fly to be added to the assay is mixed first in I x incubation buffer with 1 mM DTF. For peptide competition experiments we usually test the peptide at various concentration in the presence and absence of 50-100 nM fly. So we make up two solutions, and one that contains 6x f ) , (600 nM for a final of 100 nM) and one containing an equivalent volume of buffer that the fly was stored in. For example, if we have 33/zM f ) , stock and we want 150/zl of 600 nM fly

[39]

LIGANDSFOR/3y SUBUNITSFROM PEIrI'IDELIBRARIES

575

we would need to add 75/zl 2x Inc. Buffer, 8/zl/3y, 1.5/zl 0.1 M DTF, 65.5/zl water. The solution without/3y is the same except 8/zl of 13y storage buffer would be added instead of/3y. If the/3y needs to be diluted before it is added to the incubation buffer, this should be done in/~ ~, storage solution. Peptide Solutions. Peptides are diluted in water to 6 times the desired final concentrations. The stock peptide solutions are made at 1-5 mM and the concentration verified using the peptide bond absorbance at 215 nm. We usually buy the peptides at 80-95% purity. If the peptide needs to be oxidized for activity the peptide is dissolved at 1 mg/ml, the pH of the peptide solution is adjusted to pH 7-8, and the solution is incubated overnight at room temperature with exposure to air. Ca 2+ Solutions. Make up 1 ml of fresh calcium solution every time 500/zl 2x assay buffer, 168/zl 0.1 M CaCI2, 322 #1 doubly distilled H20, 10/,tl 0.1 M DTT. The calcium can be varied depending on the pH of the reaction and the amount of final calcium concentration desired. Assay Procedure. Assays should include at least two blank reactions that contain everything except the calcium solution. The counts per minute from the blank are a measure of the [3H]PIP2 that is hydrolyzed prior to the reaction and are subtracted from all of the values obtained in the presence of Ca 2+. Aliquot 20/zl of lipid vesicle solution into 4 ml plastic reaction tube (with tubes sitting in an ice bath). Add the other reagents in the order described above including the calcium solution. To start the reaction transfer the tubes to a 30 ° water bath [leave the blanks on ice and immediately add trichloroacetic acid (TCA)]. After 5-30 min transfer the tubes back to the ice bath and add 200 #1 ice-cold 10% TCA to each tube to stop the reactions. Add 100/z110 mg/ml BSA, vortex, and centrifuge 5 min at 2000g at 4 °. Pipette out 300/zl of supernatant and transfer to a scintillation vial, add 4 ml of scintillation fluid, and analyze by liquid scintillation counting. The amount of [3H]IP3 released should not exceed 40% of the total [3H]PIP2 cpm in the assay because the assay is saturated at this point. If the counts per minute released are either too high or too low the reaction can be adjusted by changing the amount of PLC in the assay, changing the reaction time, or changing the final calcium concentration in the assay. Results of a typical peptide competition experiment are shown in Fig. 4. Summary We have described a method using polyvalent peptide display on filamentous phage that can be used to identify ligands that bind to G protein/3~/subunits. Also described is how to construct phage that have known fly binding sequences fused to the coat protein to allow a competition analysis to be performed. Once selected or constructed, these phage-beating fly-binding peptides are powerful tools for mapping interaction sites for fly binding proteins and can be used to begin to dissect the unique modes of binding for individual/3y subunit-regulated effectors.

576

G PROTEINSTRUCTUREANDFUNCTIONALDOMAINS

[39]

Appendix: Solutions for Bacterial a n d Phage M a n i p u l a t i o n Many of these solutions are take directly from Ref. 6. ABTS solution: 22 mg 2,2'-Azinobis(3-ethylbenthiazoline-6-sulfonic acid) (Sigma), 38.6 ml 0.2 M Na2HPO4, 61.4 ml 0.1 M citric acid, add 1/1000 volume of 30% (w/v) H202 before use 2% BSA: 2 g BSA in 100 ml 1× TBS NZY medium: 21 g NZY broth powder (Gibco-BRL) 1000 ml distilled H20, autoclave for 15 min. NZY agar medium: 21 g NZY broth powder, 1000 ml distilled HzO, 15 g Bacto-agar; autoclave for 15 min NZY/Tc agar medium: Autoclaved NZY agar medium, add Tc (tetracycline) at 40/zglml NZY/Tc medium: NZY medium, add Tc at 20/zg/ml PEG/NaCI: 100 g Polyethylene glycol (PEG) 8000, 116.9 g NaC1, 475 ml distilled H20, dissolve with heating if necessary TBS (10× stock): 1.5 M NaC1, 0.5 M Tris-HC1 (pH 7.5) TBS/0.1% Tween: 100 ml l x TBS, 0.1 ml Tween TBS/gelatin: 0.1% (w/v) gelatin in TBS, autoclave Tetracycline (Tc) (20-mg/ml stock): 40 mg/ml in water and filter sterilize into an equal volume of glycerol (autoclaved), store at - 2 0 ° Virus Lysis mix (5x): 2gSDS, 18 mlH20,2m140× GBB, 40mg bromphenol blue, 20 ml glycerol 100% GBB (40x stock): 142.4 g Tris, 45.94 g sodium acetate anhydrous (or 76.16 g trihydrated), 18.83 g Na2-EDTA-2H20. Adjust pH to 8.3 with acetic acid and take to final volume of 700 ml. Store at room temperature

Solutions for PLC Assay 2x Assay Buffer: 100 mM HEPES pH 7.2, 6 mM EGTA, 160 mM KC1, pH to 7.2 2x Incubation Buffer: 100 mM HEPES pH 7.2, 2 mM EDTA, 6 mM EGTA, 200 mM NaC1, 10 mM MgC12, pH to 7.2 Lipid Sonication Buffer: 750/A 2x assay buffer pH 7.2, 235/A doubly distilled H20, 15/zl 0.1 MDTT