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[11] Cell-free synthesis of peptide libraries displayed on polysomes
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[1 1] C e l l - F r e e S y n t h e s i s o f P e p t i d e L i b r a r i e s D i s p l a y e d on Polysomes By LARRY C. MATTHEAKIS, JENNIFER M. DIAS, and WILLIAM Jo DOWER
Introduction Peptide libraries displayed on phage l-3 or plasmids 4 can provide a rich source of ligands for a variety of targets including antibodies, TM enzymes,s'6 lectins,7'8 and nucleic acids. 9,1° Both display systems rely on in vivo gene expression, and the size and diversity of the library are ultimately determined by the transformation capacity and biological constraints of the Escherichia coli host. Library size becomes important when ligands of the correct structure are rare, and only a small fraction of the possible sequences can be sampled in the initial round of screening. We have developed an in vitro peptide expression system which displays the peptide library on polysomes, u This system, which avoids bacterial transformation, can generate libraries that are several orders of magnitude larger than those of cell-based systems. In addition, the diversity of sequences synthesized on polysomes may also be greater since secretion, phage assembly, and other cellular processes are not required for peptide display in vitro. A summary of the method begins with the construction of a DNA library (Fig. 1). The library consists of random peptide-coding sequences that are fused in-frame to the 5' end of a spacer sequence. The DNA library is incubated in a DNA-dependent in vitro transcription/translation system, and polysomes are isolated by high-speed centrifugation. The polysomes, i S. E. Cwirla, E. A. Peters, R. W. Barrett, and W. J. Dower, Proc. Natl. Acad. Sci. U.S.A. 87, 6378 (1990). 2 j. j. Devlin, L. C. Panganiban, and P. E. Devlin, Science 249, 404 (1990). 3 j. K. Scott and G. P. Smith, Science 249, 386 (1990). 4 M. G. Cull, J. F. Miller, and P. J. Schatz, Proc. Natl. Acad. Sci. U.S.A. 89, 1865 (1992). 5 D. J. Matthews and J. A. Wells, Science 260, 1113 (1993). 6 p. j. Schatz, Bio/Technology 11, 1138 (1993). 7 K. R. Oldenburg, D. Loganathan, I. J. Goldstein, P. G. Schultz, and M. A. Gallop, Proc. Natl. Acad. Sci. U.S.A. 89, 5393 (1992). s j. K. Scott, D. Loganathan, R. B. Easley, X. Gong, and I. J. Goldstein, Proc. Natl. Acad. Sci. U.S.A. 89, 5398 (1992). '~A. C. Jamieson, S.-H. Kim, and J. A. Wells, Biochemistry 33, 5689 (1994). l0 E. J. Rebar and C. O. Pabo, Science 263, 671 (1994). u L. C. Mattheakis, R. R. Bhatt, and W. J. Dower, Proc. Natl. Acad. Sci. U.S.A. 91, 9022 (1994).
METHODS IN ENZYMOLOGY, VOL. 267
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. An in vitro polysome system for screening peptide libraries. (1) A synthetic DNA library containing a random NNK codon region is incubated in an E. coli $30 coupled transcription/translation system. (2) Protein synthesis is stopped with chloramphenicol,and polysomes are isolated by centrifugation. (3) Polysomes are added to wells containing an immobilized receptor for affinity selection. (4) The bound polysomes are dissociated with EDTA and mRNA is recovered. (5) mRNA is copied into cDNA. (6) cDNA is amplified by PCR using primers that restore the T7 promoter (Pw). (7) A portion of the enriched pool is cloned into a phagemid or MBP vector for ELISA and sequencing before repeating the cycle. (Reprinted from Mattheakis et al., 11 with permission).
consisting of nascent peptides linked to their encoding mRNAs, are screened by affinity selection of the nascent peptides on an immobilized target. The polysome-bound m R N A is recovered, copied onto cDNA, and amplified by polymerase chain reaction (PCR) to produce template for the next round of i n v i t r o synthesis and selection. After each round, the enriched peptides are identified by cloning and sequencing a portion of the amplified template and their binding specificities are determined using various assays. The following describes these steps in detail and discusses applications of the technology to ligand discovery.
C o n s t r u c t i o n of DNA Library The D N A library genes are designed for high-level expression of nascent peptide~. Each library m e m b e r is under the transcriptional control of the bacteriophage T7 p r o m o t e r and uses the T7 gene 10 ribosome-binding site. The coding sequence of the library consists of random peptide sequences that are fused in-frame to a constant spacer sequence. The function of the
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CCAGGGCGTTGGTGAATTCTCCGGCAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTTCCGG Q G V G E F S G S G S G S G S G S G S G CAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTGGATCCCAGTCGGTTGA S G S G S G S G S G S G S G G S Q S V E ATGTCGCCCTTATGTCTTTGGCGCTGGTAAACCATATGAAT%~TCTATTGATTGTGACAA C R P Y V F G A G K P Y E F S I D C D K AATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGT I N L F R G V F A F L L Y V A T F M Y V ATTTTC GACGTTTGCTAACATAC TGTCGACAGAAGGAGAAGGAGAAGGAGAAGGAGAAGG F S T F A N I L S T E G E G E G E G E G AGAAGGACGACAGGCGACACGCAGGCAGCAGGCGCCAAGCTTGTCACATGCACGATCCTC E G R Q A T R R Q Q A P S L S H A R S S CAATTCCACAATCGTGG N S T I V
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197 20 40 60 80 i00 120 125
FIG. 2. Nucleotide sequence and predicted amino acid sequence of the 377-bp spacer fragment isolated from plasmid pLM182. Nucleotides are numbered on the left and amino acids are numbered on the right. The plII-derived sequence begins at amino acid position 37 and ends at position 88. The BstXI sites are underlined.
spacer sequence is twofold: to provide a flexible linker for the nascent peptide and to slow the rate of translation termination by including rare codons, m R N A secondary structures, or other sequences that result in ribosome stalling. Our spacer, which is modified from a previous study, H encodes a linker composed of alternating Gly-Ser residues and a stalling sequence that includes a segment from the structural gene of the bacteriophage plII protein (Fig. 2). The D N A library is constructed by in vitro ligation. The spacer fragment, flanked by noncomplementary BstXI sites, is isolated from plasmid pLM182 by BstXI digestion and is ligated to a fragment encoding the T7 promoter and random peptide sequences. To construct the random peptide D N A fragment, two oligonucleotides are synthesized: one encoding the T7 promoter and gene I0 ribosome-binding site, and the other encoding a degenerate codon region of the form NNK, where N is equimolar A, C, G, or T and K is G or T. There are 32 possible codons resulting from the NNK motif: 1 for each of 12 amino acids, 2 for each of 5 amino acids, 3 for each of 3 amino acids, and 1 stop codon (amber). Both oligonucleotides share a short region of complementary sequence which is used for annealing and subsequent extension by D N A polymerase. The BstXI site at the 3' end of the random peptide fragment can only ligate to the BstXI site at the 5' end of the spacer fragment, thus ensuring directional ligation. The efficiency of oligonucleotide synthesis limits this procedure to libraries containing less than 30 random amino acids. For longer sequences, it should be possible to synthesize multiple fragments and join them by other methods such as overlap extension. 12 12 R. M. Horton, H. D. Hunt, S. N. Ho, J. K. Pullen, and L. R. Pease, Gene 77, 61 (1989).
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Materials ON1543: 5' ACT TCG A A A TTA ATA CGA CTC ACT A T A G G G A G A CCA C A A CGG TTT CCC TCT A G A A A T A A T TTT G T r T A A CTT T A A G A A G G A GAT A T A CAT 3'. The T7 promoter is underlined. ON1747: 5' A A ATT TCC A A C GCC CTG GGT ACC (MNN)10 GCT A G C CAT A T G TAT ATC TCC TTC TT 3'. This particular library encodes a random 10-mer peptide. An M base is C or A and is complementary to K (G or T). The BstXI site used for ligation is underlined. 10x extension buffer: 400 mM Tris-HC1 (pH 7.5), 50 m M MgCI2, 20 m M dithiothreitol (DTT), 500 mM NaCI, 500/zg/ml bovine serum albumin (BSA), and 3 mM each of dATP, dCTP, dGTP, and dTTP. Sequenase version 2.0, 13 units//A (USB Biochemicals, Cleveland, OH). Spin columns: MicroSpin S-200 H R columns (Pharmacia Biotech, Piscataway, N J). Gel solubilization buffer: 6 M NaI, 50 m M Tris-HCl (pH 8.0), 0.05% (w/v) Na2SO3, and 10 mM CDTA (1,2-cyclohexanediaminetetraacetic acid). Low melting agarose: NuSieve GTG agarose (FMC Bioproducts, Rockland, ME). Wizard PCR preps D N A purification system (Promega Corp., Madison, WI).
Procedure for Constructing DNA Library Anneal 100 pmol each of oligonucleotides ON1543 and ON1747 in a final volume of 28/zl containing 3 tzl of ]0X extension buffer. Add 2/zl of Sequenase and incubate at 37° for 1 hr. Add 40/zl of water, 5/A of BstXI enzyme (50 units), and 5/xl of 10x BstXI enzyme buffer. Incubate at 55 ° for several hours or overnight. Inactivate the BstXI enzyme by heating the reaction to 65° for 20 min, and desalt the mixture on a S-200 spin column. Store at - 2 0 °. To isolate the spacer fragment, cut plasmid pLM182 with BstXI and gel purify the 377-bp fragment. Alternatively, the fragment can be isolated by PCR amplification. Since several micrograms of the spacer fragment are required for ligation, it is important to have an efficient gel purification method. We use a 3% NuSieve GTG agarose gel. The gel slices are dissolved by adding 2 volumes of gel solubilization buffer and heating to 55 ° until the slices have completely melted; the D N A is recovered using the Wizard PCR gel purification system.
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To ligate, set up several reaction tubes, each containing 1 /zg of the spacer fragment and a fourfold molar excess of the random peptide DNA fragment. Add ligase buffer, 600 units of ligase (New England Biolabs), and water to a final volume of 25/.d. Incubate overnight at 14°. Check the efficiency of ligation by running a small aliquot of the ligated mixture on a gel alongside the purified random peptide and spacer DNA fragments. Under these conditions, nearly all of the spacer fragment is converted to the ligated product (509 bp for a random 10-mer library). Gel purify the ligated fragment using the Wizard system and determine its concentration. Store at - 2 0 °. In Vitro Peptide Synthesis and Polysome Screening
All of our polysome screening studies use the E. coli S30-coupled transcription/translation system. We chose the $30 system because it translates mRNA with high efficiency, is a simpler system (requiring fewer translation factors) than rabbit reticulocyte or wheat germ, and is capable of coupled transcription/translation which avoids the necessity of carrying out a separate transcription reaction of the DNA library. The $30 system contains all of the components required for DNAdependent in vitro translation of E. coli genes. We found that a commercially available $30 system from Promega works well. The Promega extract is prepared from an E. coli strain that is deficient in omp T endoproteinase, Ion protease, and exonuclease V, which together reduce the degradation of linear templates and increase the stability of expressed peptides. Because our DNA library is under the transcriptional control of the T7 promoter, we supplement the $30 system with purified T7 RNA polymerase and rifampicin to inhibit the endogenous E. coli RNA polymerase and ensure that only the DNA library will be transcribed. Approximately 400 ng (1012 molecules) of DNA library can be added to the in vitro system without saturating the transcriptional or translational capacity of a 50-bd reaction. H The reactions are incubated until the rate of protein synthesis has reached steady state and are stopped by adding chloramphenicol, which binds tightly to the 50S ribosomal subunit to inhibit elongation and stabilize the polysome complex. Under these conditions, about 3 mol of mRNA is synthesized per mole of DNA, and approximately 30% of the mRNA pool (1012 mRNA molecules) is bound specifically to ribosomes. The polysome complexes are then isolated by centrifugation, and the nascent peptides are screened by affinity selection. To recover the polysome-bound mRNA, EDTA is added to chelate Mg 2÷ ions and dissociate the ribosomal subunits. The EDTA elution step is specific and recovers the bound mRNA without disrupting the peptide-target complex.
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Because these reaction conditions are amenable to scale up, it should be possible to screen even larger libraries (1013 to 10TM DNA molecules) by increasing the $30 reaction volume 10- to 100-fold. Materials
Polysome buffer: 20 mM HEPES-KOH, pH 7.5, 10 mM MgC12, 15 /xg/ml chloramphenicol, 100/zg/ml ~cetylated (BSA), 0.1% Tween 20. The HEPES and MgCI2 solutions are prepared separately, treated with 0.1% diethyl pyrocarbonate, and autoclaved to remove any trace amounts of ribonuclease. In previous studies, we included RNasin (Promega) and DTT in the polysome and elution buffers11; however, we found that these reagents can be omitted from the binding and elution steps without affecting the stability of the mRNA. 13 Elution buffer: The elution buffer is polysome buffer lacking Tween 20, but including 20 mM EDTA. E. coli $30 extract translation system for linear templates (Promega): This system includes an E. coli $30 extract and a complete premix that includes nucleotides, tRNAs, an ATP-regenerating system, and all 20 amino acids. Several methods have been published for preparing the $30 extract and reaction buffers. 14-16 T7 RNA polymerase: 200 units//zl (Ambion, Austin, TX). Rifampicin: 1 mg/ml in 10% (v/v) methanol (Boheringer Mannheim, Indianapolis, IN). Borate buffer: Dissolve 30.9 g of H3BO3 in 900 ml of water. Adjust the pH to 9.5 using 5 M NaOH and bring the final volume to 1 liter with water. PBT buffer: PBS (140 mM NaC1, 3 mM KC1, 2 mM K3PO4, 10 mM NaH2PO4) containing 0.1% BSA (fraction V), 0.1% Tween 20, and 0.02% NAN3. Tosyl-activated M-450 magnetic beads (Dynal, Great Neck, NY). Procedure
On ice, combine 20/zl of complete premix, 15/zl of $30 extract, 1/zl of T7 RNA polymerase, 1 /xl of rifampicin, and at least 400 ng of DNA library. Add nuclease-free water to bring the final volume to 50/zl and incubate the reactions at 37° for 30 min. To stop the reaction, place the 13j. M. Dias and L. C. Mattheakis,unpublishedobservations (1995). 14S. A. Lesley,M. A. D. Brow, and R. R. Burgess,J. BioL Chem. 266, 2632 (1991). 15j. M. Pratt, in "Transcriptionand Translation:A PracticalApproach" (B. D. Haines and S. J. Higgins,eds.), pp. 179-209. IRL Press, Oxford, 1984. ~6G. Zubay,Annu. Rev. Genet. 7, 267 (1973).
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tube on ice and add 150 t.d of cold polysome buffer. Transfer the diluted reaction to a polycarbonate tube and centrifuge in a Beckman TLA 100 rotor for 36 min at 90,000 rpm at 4°. Promptly remove the supernatant, making sure to remove all traces of residual liquid. Resuspend the gelatinous polysome pellet in 300/zl of polysome buffer by gently pipetting up and down, and transfer to a microcentrifuge tube. Incubate the tube, with slow end-over-end rotation, for 30 min at 4°. Centrifuge at 10,000 g for 5 min to remove any insoluble material, transfer the cleared polysome supernatant to a fresh tube, and store on ice. The screening target can be immobilized on microtiter wells or magnetic beads. We found that nonspecific binding of polysomes is lower when beads are used. 13 Proteins, such as antibodies, can be chemically conjugated to tosyl-activated beads, and biotinylated nucleic acids can be attached to beads that have been coated with streptavidin. Receptors, containing an epitope tag, can be immobilized to beads conjugated to a nonblocking monoclonal antibody (MAb). To immobilize a MAb, dilute it to 150/xg/ml in borate buffer and add an equal volume of bead suspension (30 mg/ml). Incubate overnight at room temperature with slow end-over-end rotation and collect the beads by magnetic separation or by low-speed centrifugation. Discard the supernatant and resuspend the beads in 500/zl of PBT buffer. Incubate for 10 min and repeat the washing step three times. Incubate the fourth wash overnight at 4° with slow end-over-end rotation. Discard the supernatant and resuspend the beads in PBT buffer to a final bead concentration of 15 mg/ml. The MAb-conjugated beads have a shelf-life of at least 3 months when stored at 4°. All of the binding and washing steps are done at 4°. To bind polysomes, add 10/zl of MAb-conjugated beads (equivalent to 150/zg beads or about 106 bead particles) to 300/zl of the cleared polysome solution and incubate for 2 hr with slow end-over-end rotation. Collect the beads by magnetic separation and discard the supernatant. Gently wash the beads five times with 200/xl of polysome buffer. After the final wash, add 100 tzl of elution buffer and incubate for an additional 10 min at 4° with rotation. Collect the beads and save the supernatant containing the eluted mRNA. Store the eluted mRNA at -20 °. cDNA Synthesis and PCR Amplification Primers
ON1914: 5' GAT TGT G G A AGC TlCG GCG CCT GCT 3' ON1230: 5' GGC GCC TGC TGC CTG CGT GTC GCC TGT CGT 3' ON2856: 5' CGA AAT TAA TAC GAC TCA CTA TAG GGA GAC CAC AAC GGT TTC CCTC 3'
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Procedure The eluted m R N A is treated with DNase I to remove any D N A template that may have carried over from the transcription step. Because the elution buffer contains 20 m M EDTA, the sample must be supplemented with MgCI2 for DNase I activity. Add 4/xl of 1 M MgCI2 (final concentration of 40 mM) and 4 units of RNase-free DNase I (Ambion). Incubate at 37° for 15 min. Extract with 100/xl of phenol and ethanol precipitate the mRNA in the presence of 10/xg of glycogen (Boehringer Mannheim, molecular biology grade). Wash the pellet with 70% (v/v) ethanol and resuspend it in 17/zl of nuclease-free water. To maximize the recovery of sequences, all of the eluted mRNA recovered after the first round of screening is copied into cDNA. For subsequent rounds, half of the m R N A is saved in case the cDNA synthesis or PCR amplification steps need to be repeated. For cDNA synthesis, heat 8.5/zl of the m R N A to 75° for 3 min. Place the tube on ice and add the following reagents: 2/.d of primer ON1914 (25/zM), 4/xl of MgC12 (25 raM), 2/zl of 10× RT buffer (100 m M Tris-HC1, pH 8.8, 500 mM KCI, 1% Triton X-100), 2/xl of 10 m M each dNTP, 0.5/.d (20 units) of recombinant RNasin (Promega), and 1/zl (20 units) of avian myeloblastosis virus reverse transcriptase (Promega). Incubate at 42 ° for 40 rain. Precipitate the cDNA with ethanol as described earlier and resuspend the pellet in 11/xl of water. Prepare 10 PCR reactions containing 20 mM Tris-HC1 (pH 8.4), 50 mM KCI, 1 mM MgCl2, 0.5 /.~M each of ON2856 and ON1230, 10% glycerol, 0.2 mM each dNTP, and 1/zl of cDNA in a final volume of 100 /M. Cover the tubes with mineral oil and heat them for 2 min at 94°. Lower the temperature to 72 ° and add 0.5/zl (2.5 units) of Taq D N A polymerase for a "hot start." Incubate for 30 cycles at 94 ° for 30 sec, 50° for 30 sec, and 72 ° for 45 sec. Combine the PCR reactions and precipitate the amplified template with ethanol. Resuspend the D N A in 25 /zl of water and purify it by electrophoresis on a 3% NuSieve GTG agarose gel. Excise the 472-bp template band and recover the D N A using the Wizard system (Promega) as described previously for constructing the D N A library. The amplified template band is slightly smaller than the starting library because the primer ON1230 hybridizes 37 bp upstream from the 3' BstXI site of the spacer fragment.
Cloning and Sequencing of Enriched Sequences and Determining Peptide Binding Specificities To identify individual sequences that have been enriched during selection, it is necessary to clone and sequence the pools of amplified D N A
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FI~. 3. Cloning vectors for sequencing and ELISA of enriched sequences. The partial nucleotide and predicted amino acid sequences of the amplifiedDNA library pool are shown on the top. The partial sequences for the plII phagemid vector pAFF6 (the arrow indicates the processing site for signal peptidase), MBP vector pLM190, and the polysome expression plasmid pLM193 are shown below. The NheI and KpnI cloning sites are underlined.
after each round. We have constructed cloning vectors that express the enriched peptide sequences either as N-terminal fusions to the phage coat protein pIII or as C-terminal fusions to the maltose-binding protein (MBP) (Fig. 3). The fusions are tested for binding activity using an ELISA, and the peptide-coding regions of the positive clones are sequenced. The phage E L I S A is also a convenient method for determining enrichment during polysome screening. Pools of sequences from each round of polysome screening are cloned into the plII phagemid vector, pAFF6, and a phage supernatant is prepared from the pool of transformants. If the phage pool is positive in the E L I S A , then individual clones from the same transformation mixture are tested for binding activity and sequenced. The MBP vector, pLM190, is used for expressing the peptides as Cterminal fusions to a cytoplasmic form of MBP (Fig. 3). Switching the flee end to C-terminal display can potentially affect binding activity, but there are several reasons for using an MBP ELISA. First, the MBP E L I S A is more affinity sensitive than the phage E L I S A , permitting a ranking of related sequences according to their affinities. 17 Second, for more detailed studies such as competition-binding assays, which require purified fusion protein, MBP can be expressed in large amounts as a soluble protein and is easily purifiedJ 8 iv C. M. Gates, E. L, Martin, and P. J. Schatz, personal communication (1995). 18p. j. Schatz, M. G. Cull, E. L. Martin, and C. M. Gates, Methods Enzymol. 267, Chap. 10, 1996 (this volume).
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Expressing a peptide fused to plII or MBP in E. coli is convenient for determining its binding specificity by ELISA, but may introduce biological biases which mask the binding activity that was selected for in vitro. For this reason, we have also developed an in vitro polysome-binding assay. 11 In this assay, a D N A template encoding the sequence to be tested is incubated in an in vitro reaction that is supplemented with radiolabeled UTP to label the newly synthesized mRNA. Polysomes, radiolabeled by virtue of the bound mRNA, are isolated and added to the immobilized target. After binding and washing, E D T A is added to dissociate the bound complexes and the radiolabeled m R N A is recovered and counted. In general, for a new target, we prefer the convenience of the phage system for sequencing and ELISA. If the sequence analysis reveals a strong consensus sequence that is negative in both the phage and MBP ELISA, then the polysome-binding assay is a useful alternative for determining the specificity of binding.
Procedure for Phage ELISA and Sequencing Digest 500 ng of the amplified D N A with NheI and KpnI. Extract once with phenol and three times with ether to remove any residual phenol. We avoid precipitating the digested library with ethanol because the random peptide-coding fragments are small (less than 60 bp) and do not precipitate efficiently. To ligate the fragments to pAFF6, combine 250 ng of the digested library with ligase buffer, 400 units of ligase, and 50 ng of vector that has been cut with NheI/KpnI and gel purified by the Geneclean method (Bio 101). Incubate overnight at 14°. Precipitate the ligated mixture with ethanol in the presence of glycogen as described earlier. Wash the pellet with 70% ethanol and resuspend it in 10/zl of water. Remove 5/zl and transform 20 /zl of E. coli strain XL1-Blue (Stratagene, San Diego, CA) by electroporation using SOC medium for the outgrowth step. 19 Grow the transformants for 1 hr at 37 ° without shaking, and plate 0.1 ml of undiluted to 10 -2 dilutions on LB-ampicillin (100/zg/ml) plates. Colonies from these plates are used to sequence and determine the binding specificity of the individual clones. To prepare a phage supernatant of the transformed pool, add 0.5 ml of the outgrown cells to 10 ml of phagemid growth medium (LB containing 0.25% K H 2 P O 4 , 0 . 1 % each of MgSO4 and glucose, and 100/.~g/ml of ampicilfin) and incubate overnight at 37°. Add 0.5 ml of the overnight culture to 10 ml of fresh phagemid growth medium and grow until the A600 reaches 0.6 to 0.8. Remove 1 ml of cells and infect with 5 x 10 9 plaque-forming units of VCSM13 helper phage (Stratagene) per ml of cells. Incubate at 19W. J. Dower, J. F. Miller, and C. W. Ragsdale, Nucleic Acids Res. 16, 6127 (1988).
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37° for 30 min without shaking to allow for attachment of the helper phage. Remove 0.4 ml and add to 2 ml of phagemid growth medium supplemented with 24 tzg/ml kanamycin and 0.024% (w/v) arabinose. Grow overnight at 37° with vigorous shaking and centrifuge the cultures at 5000 g for 10 min. Store the phage supernatants at 4° for 1 to 2 days or at - 2 0 ° for longer periods. To prepare a phage supernatant of individual clones, inoculate each colony in 5 ml of phagemid growth medium. Grow at 37 ° for 4 hr with vigorous shaking. Infect with VCSM13 helper phage as described earlier and add kanamycin and arabinose to a final concentration of 20/zg/ml and 0.02%, respectively. Grow overnight at 37°, centrifuge the culture, and store the phage supernatant at 4°. For the phage ELISA, prepare a polystyrene 96-well microtiter plate (Immulon 4, Dynatech). Each phage supernatant requires four wells: two are coated with the target and two are left as blanks. The concentration of target added to the wells must be optimized on a case-by-case basis. For a MAb, add 1 to 5/xg in 100/xl of PBS to each well. Incubate the plate at 37 ° for 1 hr. Wash the wells five times with PBS and block each well with 250 tzl of PBS/I% BSA. Incubate the plate at 37° for 1 hr and wash again five times with PBS. To each well, add 50/zl of PBS/0.1% BSA and 50/~1 of the phage supernatant. Incubate for 2 hr at 4°, shaking gently on a microtiter plate shaker. Wash the wells six times with PBS and add 50 t~l of horseradish peroxidase-conjugated to sheep anti-M13 IgG (Pharmacia) that has been diluted 1 : 5000 in PBS/1% BSA. Incubate for 1 hr at 4 ° on the plate shaker and wash the wells six times with PBS. Binding is detected by adding 100/xl of substrate [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)diammonium (0.2 mg/ml), 50 mM citric acid, pH 4/0.05% (v/v) hydrogen peroxide] and measuring the A405. Phage binding is scored as positive if the average A405 for the duplicate wells coated with the MAb is at least twofold greater than that of the corresponding control wells. To sequence the phage clones, prepare phagemid D N A from the phage supernatants using the Prep-A-Gene system (Bio-Rad). The primer ON-3 (5' CGATCTAAAGTTTTGTCGTCT 3') is a reverse sequencing primer that hybridizes 80 bp downstream from the KpnI site of pAFF6.
Procedure for MBP ELISA To clone into the MBP vector pLM190, add 50 ng of the cut and gelpurified vector to 250 ng of the NheI/KpnI cut amplified D N A pool. Ligate as described earlier and transform E. coli strain ARI 814 ~s by electroporation to isolate individual ampicillin-resistant colonies. Follow the steps for the MBP ELISA. is
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Procedure for Polysome Binding Assay Ligate an individual sequence or a pool of amplified D N A sequences into the NheI/KpnI sites of pLM193. Electroporate E. coli strain XL1Blue and isolate individual ampicillin-resistant colonies as described earlier. Inoculate each individual colony into 10 ml of LB-ampicillin (amp), grow overnight at 37 °, and isolate plasmid D N A using the Qiagen plasmid purification system (Qiagen Corp., Chatsworth, CA). Linearize the plasmid by cutting 5 tzg with PacI, which cleaves just downstream of the 3' end of the spacer sequence. Extract the cut plasmid with phenol and precipitate it with ethanol. Resuspend the pellet in nuclease-free water to a concentration of about 250 ng//xl. Add 1/xg of the PacI-cleaved plasmid D N A encoding each sequence to be tested and a negative control, nonbinding, sequence to separate in vitro reactions supplemented with 10/zCi [ot-33p]UTP(Amersham, Arlington Heights, IL, 3000 Ci/mmol). Incubate the reactions and isolate the polysomes as described previously for polysome screening. Remove two 1-/M aliquots of the cleared polysome solution and precipitate each in 1 ml of trichloroacetic acid (TCA) that contains 25/xg of acetylated BSA. Collect the TCA precipitates on GF/C glass fiber filter disks (Whatman, Clifton, NJ) and count them in a liquid scintillation counter. Determine the average counts per minute (cpm) for each set of duplicates and use this value to normalize the polysome input for each sequence. Add approximately 100,000 cpm of the polysome preparation to the binding reaction and elute the m R N A as described previously. Precipitate, using TCA, two 40-/zl aliquots of the eluted m R N A and determine the average cpm that was recovered. If binding is specific, the cpm corresponding to the test sequence should be at least threefold greater than that obtained for the negative control sequence. Alternatively, the negative control can be the same polysome preparation added to beads preblocked with a competing ligand. Concluding Remarks The polysome display system is a recent biological method for screening peptide libraries. Unlike the cell-based methods, polysome screening relies on in vitro synthesis and amplification of the peptide sequences during each round of screening. This offers the potential of screening larger libraries and a more diverse collection of sequences since few biological biases should affect gene expression in vitro. It should also be possible to expand the structures of amino acids comprising the library by supplementing the in vitro system with suppressor tRNAs that have been chemically acylated with unnatural amino acids, a° 20C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, and P. G. Schultz,Science244, 182 (1989).
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A notable difference between phage display and the current polysome display system is the affinities of the recovered peptides. We find that highaffinity sequences (Kd values of less than 200 nM) predominate the pools of enriched sequences, which suggests that polysome display may be monovalent. This is in contrast to the multivalent phage and plasmid display systems which can recover ligands with affinities as low as 100/xM. 21Monovalent peptide display limits the range of ligand families that are recovered, but is very effective in selecting higher affinity sequences, especially if mutagenic PCR 22 is incorporated between rounds of selection. Repeated rounds of polysome screening result in certain sequences dominating the population, and often the most frequently occurring sequence will bind to the target with the highest affinity. For screening random libraries, it would be desirable to take advantage of the enormous diversity of sequences displayed on polysomes and enrich for lower affinity ligands. This could be accomplished by increasing the peptide valency by modifying the spacer sequence to increase the frequency of ribosome stalling. We estimate that an efficient stalling sequence at the 3' end of the spacer could result in up to five stalled ribosomes displaying the peptide. Such modifications of the spacer sequence should lead to further improvement and utility of the polysome display system.
21 E. M. Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor, and M. A. Gallop, J. Med. Chem. 37, 1385 (1994). 22 R. C. Cadwell and G. F. Joyce, PCR Methods Appl. 2, 28 (1992).
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[1 1] C e l l - F r e e S y n t h e s i s o f P e p t i d e L i b r a r i e s D i s p l a y e d on Polysomes By LARRY C. MATTHEAKIS, JENNIFER M. DIAS, and WILLIAM Jo DOWER
Introduction Peptide libraries displayed on phage l-3 or plasmids 4 can provide a rich source of ligands for a variety of targets including antibodies, TM enzymes,s'6 lectins,7'8 and nucleic acids. 9,1° Both display systems rely on in vivo gene expression, and the size and diversity of the library are ultimately determined by the transformation capacity and biological constraints of the Escherichia coli host. Library size becomes important when ligands of the correct structure are rare, and only a small fraction of the possible sequences can be sampled in the initial round of screening. We have developed an in vitro peptide expression system which displays the peptide library on polysomes, u This system, which avoids bacterial transformation, can generate libraries that are several orders of magnitude larger than those of cell-based systems. In addition, the diversity of sequences synthesized on polysomes may also be greater since secretion, phage assembly, and other cellular processes are not required for peptide display in vitro. A summary of the method begins with the construction of a DNA library (Fig. 1). The library consists of random peptide-coding sequences that are fused in-frame to the 5' end of a spacer sequence. The DNA library is incubated in a DNA-dependent in vitro transcription/translation system, and polysomes are isolated by high-speed centrifugation. The polysomes, i S. E. Cwirla, E. A. Peters, R. W. Barrett, and W. J. Dower, Proc. Natl. Acad. Sci. U.S.A. 87, 6378 (1990). 2 j. j. Devlin, L. C. Panganiban, and P. E. Devlin, Science 249, 404 (1990). 3 j. K. Scott and G. P. Smith, Science 249, 386 (1990). 4 M. G. Cull, J. F. Miller, and P. J. Schatz, Proc. Natl. Acad. Sci. U.S.A. 89, 1865 (1992). 5 D. J. Matthews and J. A. Wells, Science 260, 1113 (1993). 6 p. j. Schatz, Bio/Technology 11, 1138 (1993). 7 K. R. Oldenburg, D. Loganathan, I. J. Goldstein, P. G. Schultz, and M. A. Gallop, Proc. Natl. Acad. Sci. U.S.A. 89, 5393 (1992). s j. K. Scott, D. Loganathan, R. B. Easley, X. Gong, and I. J. Goldstein, Proc. Natl. Acad. Sci. U.S.A. 89, 5398 (1992). '~A. C. Jamieson, S.-H. Kim, and J. A. Wells, Biochemistry 33, 5689 (1994). l0 E. J. Rebar and C. O. Pabo, Science 263, 671 (1994). u L. C. Mattheakis, R. R. Bhatt, and W. J. Dower, Proc. Natl. Acad. Sci. U.S.A. 91, 9022 (1994).
METHODS IN ENZYMOLOGY, VOL. 267
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Clone and Sequence
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FIG. 1. An in vitro polysome system for screening peptide libraries. (1) A synthetic DNA library containing a random NNK codon region is incubated in an E. coli $30 coupled transcription/translation system. (2) Protein synthesis is stopped with chloramphenicol,and polysomes are isolated by centrifugation. (3) Polysomes are added to wells containing an immobilized receptor for affinity selection. (4) The bound polysomes are dissociated with EDTA and mRNA is recovered. (5) mRNA is copied into cDNA. (6) cDNA is amplified by PCR using primers that restore the T7 promoter (Pw). (7) A portion of the enriched pool is cloned into a phagemid or MBP vector for ELISA and sequencing before repeating the cycle. (Reprinted from Mattheakis et al., 11 with permission).
consisting of nascent peptides linked to their encoding mRNAs, are screened by affinity selection of the nascent peptides on an immobilized target. The polysome-bound m R N A is recovered, copied onto cDNA, and amplified by polymerase chain reaction (PCR) to produce template for the next round of i n v i t r o synthesis and selection. After each round, the enriched peptides are identified by cloning and sequencing a portion of the amplified template and their binding specificities are determined using various assays. The following describes these steps in detail and discusses applications of the technology to ligand discovery.
C o n s t r u c t i o n of DNA Library The D N A library genes are designed for high-level expression of nascent peptide~. Each library m e m b e r is under the transcriptional control of the bacteriophage T7 p r o m o t e r and uses the T7 gene 10 ribosome-binding site. The coding sequence of the library consists of random peptide sequences that are fused in-frame to a constant spacer sequence. The function of the
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PEPTIDE LIBRARIES DISPLAYED ON POLYSOMES
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CCAGGGCGTTGGTGAATTCTCCGGCAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTTCCGG Q G V G E F S G S G S G S G S G S G S G CAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTTCCGGCAGCGGTGGATCCCAGTCGGTTGA S G S G S G S G S G S G S G G S Q S V E ATGTCGCCCTTATGTCTTTGGCGCTGGTAAACCATATGAAT%~TCTATTGATTGTGACAA C R P Y V F G A G K P Y E F S I D C D K AATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGT I N L F R G V F A F L L Y V A T F M Y V ATTTTC GACGTTTGCTAACATAC TGTCGACAGAAGGAGAAGGAGAAGGAGAAGGAGAAGG F S T F A N I L S T E G E G E G E G E G AGAAGGACGACAGGCGACACGCAGGCAGCAGGCGCCAAGCTTGTCACATGCACGATCCTC E G R Q A T R R Q Q A P S L S H A R S S CAATTCCACAATCGTGG N S T I V
61 121 181 241 301 361
197 20 40 60 80 i00 120 125
FIG. 2. Nucleotide sequence and predicted amino acid sequence of the 377-bp spacer fragment isolated from plasmid pLM182. Nucleotides are numbered on the left and amino acids are numbered on the right. The plII-derived sequence begins at amino acid position 37 and ends at position 88. The BstXI sites are underlined.
spacer sequence is twofold: to provide a flexible linker for the nascent peptide and to slow the rate of translation termination by including rare codons, m R N A secondary structures, or other sequences that result in ribosome stalling. Our spacer, which is modified from a previous study, H encodes a linker composed of alternating Gly-Ser residues and a stalling sequence that includes a segment from the structural gene of the bacteriophage plII protein (Fig. 2). The D N A library is constructed by in vitro ligation. The spacer fragment, flanked by noncomplementary BstXI sites, is isolated from plasmid pLM182 by BstXI digestion and is ligated to a fragment encoding the T7 promoter and random peptide sequences. To construct the random peptide D N A fragment, two oligonucleotides are synthesized: one encoding the T7 promoter and gene I0 ribosome-binding site, and the other encoding a degenerate codon region of the form NNK, where N is equimolar A, C, G, or T and K is G or T. There are 32 possible codons resulting from the NNK motif: 1 for each of 12 amino acids, 2 for each of 5 amino acids, 3 for each of 3 amino acids, and 1 stop codon (amber). Both oligonucleotides share a short region of complementary sequence which is used for annealing and subsequent extension by D N A polymerase. The BstXI site at the 3' end of the random peptide fragment can only ligate to the BstXI site at the 5' end of the spacer fragment, thus ensuring directional ligation. The efficiency of oligonucleotide synthesis limits this procedure to libraries containing less than 30 random amino acids. For longer sequences, it should be possible to synthesize multiple fragments and join them by other methods such as overlap extension. 12 12 R. M. Horton, H. D. Hunt, S. N. Ho, J. K. Pullen, and L. R. Pease, Gene 77, 61 (1989).
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LIBRARIESON POLYSOMES
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Materials ON1543: 5' ACT TCG A A A TTA ATA CGA CTC ACT A T A G G G A G A CCA C A A CGG TTT CCC TCT A G A A A T A A T TTT G T r T A A CTT T A A G A A G G A GAT A T A CAT 3'. The T7 promoter is underlined. ON1747: 5' A A ATT TCC A A C GCC CTG GGT ACC (MNN)10 GCT A G C CAT A T G TAT ATC TCC TTC TT 3'. This particular library encodes a random 10-mer peptide. An M base is C or A and is complementary to K (G or T). The BstXI site used for ligation is underlined. 10x extension buffer: 400 mM Tris-HC1 (pH 7.5), 50 m M MgCI2, 20 m M dithiothreitol (DTT), 500 mM NaCI, 500/zg/ml bovine serum albumin (BSA), and 3 mM each of dATP, dCTP, dGTP, and dTTP. Sequenase version 2.0, 13 units//A (USB Biochemicals, Cleveland, OH). Spin columns: MicroSpin S-200 H R columns (Pharmacia Biotech, Piscataway, N J). Gel solubilization buffer: 6 M NaI, 50 m M Tris-HCl (pH 8.0), 0.05% (w/v) Na2SO3, and 10 mM CDTA (1,2-cyclohexanediaminetetraacetic acid). Low melting agarose: NuSieve GTG agarose (FMC Bioproducts, Rockland, ME). Wizard PCR preps D N A purification system (Promega Corp., Madison, WI).
Procedure for Constructing DNA Library Anneal 100 pmol each of oligonucleotides ON1543 and ON1747 in a final volume of 28/zl containing 3 tzl of ]0X extension buffer. Add 2/zl of Sequenase and incubate at 37° for 1 hr. Add 40/zl of water, 5/A of BstXI enzyme (50 units), and 5/xl of 10x BstXI enzyme buffer. Incubate at 55 ° for several hours or overnight. Inactivate the BstXI enzyme by heating the reaction to 65° for 20 min, and desalt the mixture on a S-200 spin column. Store at - 2 0 °. To isolate the spacer fragment, cut plasmid pLM182 with BstXI and gel purify the 377-bp fragment. Alternatively, the fragment can be isolated by PCR amplification. Since several micrograms of the spacer fragment are required for ligation, it is important to have an efficient gel purification method. We use a 3% NuSieve GTG agarose gel. The gel slices are dissolved by adding 2 volumes of gel solubilization buffer and heating to 55 ° until the slices have completely melted; the D N A is recovered using the Wizard PCR gel purification system.
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To ligate, set up several reaction tubes, each containing 1 /zg of the spacer fragment and a fourfold molar excess of the random peptide DNA fragment. Add ligase buffer, 600 units of ligase (New England Biolabs), and water to a final volume of 25/.d. Incubate overnight at 14°. Check the efficiency of ligation by running a small aliquot of the ligated mixture on a gel alongside the purified random peptide and spacer DNA fragments. Under these conditions, nearly all of the spacer fragment is converted to the ligated product (509 bp for a random 10-mer library). Gel purify the ligated fragment using the Wizard system and determine its concentration. Store at - 2 0 °. In Vitro Peptide Synthesis and Polysome Screening
All of our polysome screening studies use the E. coli S30-coupled transcription/translation system. We chose the $30 system because it translates mRNA with high efficiency, is a simpler system (requiring fewer translation factors) than rabbit reticulocyte or wheat germ, and is capable of coupled transcription/translation which avoids the necessity of carrying out a separate transcription reaction of the DNA library. The $30 system contains all of the components required for DNAdependent in vitro translation of E. coli genes. We found that a commercially available $30 system from Promega works well. The Promega extract is prepared from an E. coli strain that is deficient in omp T endoproteinase, Ion protease, and exonuclease V, which together reduce the degradation of linear templates and increase the stability of expressed peptides. Because our DNA library is under the transcriptional control of the T7 promoter, we supplement the $30 system with purified T7 RNA polymerase and rifampicin to inhibit the endogenous E. coli RNA polymerase and ensure that only the DNA library will be transcribed. Approximately 400 ng (1012 molecules) of DNA library can be added to the in vitro system without saturating the transcriptional or translational capacity of a 50-bd reaction. H The reactions are incubated until the rate of protein synthesis has reached steady state and are stopped by adding chloramphenicol, which binds tightly to the 50S ribosomal subunit to inhibit elongation and stabilize the polysome complex. Under these conditions, about 3 mol of mRNA is synthesized per mole of DNA, and approximately 30% of the mRNA pool (1012 mRNA molecules) is bound specifically to ribosomes. The polysome complexes are then isolated by centrifugation, and the nascent peptides are screened by affinity selection. To recover the polysome-bound mRNA, EDTA is added to chelate Mg 2÷ ions and dissociate the ribosomal subunits. The EDTA elution step is specific and recovers the bound mRNA without disrupting the peptide-target complex.
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LIBRARIESON POLYSOMES
[ 11]
Because these reaction conditions are amenable to scale up, it should be possible to screen even larger libraries (1013 to 10TM DNA molecules) by increasing the $30 reaction volume 10- to 100-fold. Materials
Polysome buffer: 20 mM HEPES-KOH, pH 7.5, 10 mM MgC12, 15 /xg/ml chloramphenicol, 100/zg/ml ~cetylated (BSA), 0.1% Tween 20. The HEPES and MgCI2 solutions are prepared separately, treated with 0.1% diethyl pyrocarbonate, and autoclaved to remove any trace amounts of ribonuclease. In previous studies, we included RNasin (Promega) and DTT in the polysome and elution buffers11; however, we found that these reagents can be omitted from the binding and elution steps without affecting the stability of the mRNA. 13 Elution buffer: The elution buffer is polysome buffer lacking Tween 20, but including 20 mM EDTA. E. coli $30 extract translation system for linear templates (Promega): This system includes an E. coli $30 extract and a complete premix that includes nucleotides, tRNAs, an ATP-regenerating system, and all 20 amino acids. Several methods have been published for preparing the $30 extract and reaction buffers. 14-16 T7 RNA polymerase: 200 units//zl (Ambion, Austin, TX). Rifampicin: 1 mg/ml in 10% (v/v) methanol (Boheringer Mannheim, Indianapolis, IN). Borate buffer: Dissolve 30.9 g of H3BO3 in 900 ml of water. Adjust the pH to 9.5 using 5 M NaOH and bring the final volume to 1 liter with water. PBT buffer: PBS (140 mM NaC1, 3 mM KC1, 2 mM K3PO4, 10 mM NaH2PO4) containing 0.1% BSA (fraction V), 0.1% Tween 20, and 0.02% NAN3. Tosyl-activated M-450 magnetic beads (Dynal, Great Neck, NY). Procedure
On ice, combine 20/zl of complete premix, 15/zl of $30 extract, 1/zl of T7 RNA polymerase, 1 /xl of rifampicin, and at least 400 ng of DNA library. Add nuclease-free water to bring the final volume to 50/zl and incubate the reactions at 37° for 30 min. To stop the reaction, place the 13j. M. Dias and L. C. Mattheakis,unpublishedobservations (1995). 14S. A. Lesley,M. A. D. Brow, and R. R. Burgess,J. BioL Chem. 266, 2632 (1991). 15j. M. Pratt, in "Transcriptionand Translation:A PracticalApproach" (B. D. Haines and S. J. Higgins,eds.), pp. 179-209. IRL Press, Oxford, 1984. ~6G. Zubay,Annu. Rev. Genet. 7, 267 (1973).
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tube on ice and add 150 t.d of cold polysome buffer. Transfer the diluted reaction to a polycarbonate tube and centrifuge in a Beckman TLA 100 rotor for 36 min at 90,000 rpm at 4°. Promptly remove the supernatant, making sure to remove all traces of residual liquid. Resuspend the gelatinous polysome pellet in 300/zl of polysome buffer by gently pipetting up and down, and transfer to a microcentrifuge tube. Incubate the tube, with slow end-over-end rotation, for 30 min at 4°. Centrifuge at 10,000 g for 5 min to remove any insoluble material, transfer the cleared polysome supernatant to a fresh tube, and store on ice. The screening target can be immobilized on microtiter wells or magnetic beads. We found that nonspecific binding of polysomes is lower when beads are used. 13 Proteins, such as antibodies, can be chemically conjugated to tosyl-activated beads, and biotinylated nucleic acids can be attached to beads that have been coated with streptavidin. Receptors, containing an epitope tag, can be immobilized to beads conjugated to a nonblocking monoclonal antibody (MAb). To immobilize a MAb, dilute it to 150/xg/ml in borate buffer and add an equal volume of bead suspension (30 mg/ml). Incubate overnight at room temperature with slow end-over-end rotation and collect the beads by magnetic separation or by low-speed centrifugation. Discard the supernatant and resuspend the beads in 500/zl of PBT buffer. Incubate for 10 min and repeat the washing step three times. Incubate the fourth wash overnight at 4° with slow end-over-end rotation. Discard the supernatant and resuspend the beads in PBT buffer to a final bead concentration of 15 mg/ml. The MAb-conjugated beads have a shelf-life of at least 3 months when stored at 4°. All of the binding and washing steps are done at 4°. To bind polysomes, add 10/zl of MAb-conjugated beads (equivalent to 150/zg beads or about 106 bead particles) to 300/zl of the cleared polysome solution and incubate for 2 hr with slow end-over-end rotation. Collect the beads by magnetic separation and discard the supernatant. Gently wash the beads five times with 200/xl of polysome buffer. After the final wash, add 100 tzl of elution buffer and incubate for an additional 10 min at 4° with rotation. Collect the beads and save the supernatant containing the eluted mRNA. Store the eluted mRNA at -20 °. cDNA Synthesis and PCR Amplification Primers
ON1914: 5' GAT TGT G G A AGC TlCG GCG CCT GCT 3' ON1230: 5' GGC GCC TGC TGC CTG CGT GTC GCC TGT CGT 3' ON2856: 5' CGA AAT TAA TAC GAC TCA CTA TAG GGA GAC CAC AAC GGT TTC CCTC 3'
202
LIBRARIES ON POLYSOMES
[ 1 11
Procedure The eluted m R N A is treated with DNase I to remove any D N A template that may have carried over from the transcription step. Because the elution buffer contains 20 m M EDTA, the sample must be supplemented with MgCI2 for DNase I activity. Add 4/xl of 1 M MgCI2 (final concentration of 40 mM) and 4 units of RNase-free DNase I (Ambion). Incubate at 37° for 15 min. Extract with 100/xl of phenol and ethanol precipitate the mRNA in the presence of 10/xg of glycogen (Boehringer Mannheim, molecular biology grade). Wash the pellet with 70% (v/v) ethanol and resuspend it in 17/zl of nuclease-free water. To maximize the recovery of sequences, all of the eluted mRNA recovered after the first round of screening is copied into cDNA. For subsequent rounds, half of the m R N A is saved in case the cDNA synthesis or PCR amplification steps need to be repeated. For cDNA synthesis, heat 8.5/zl of the m R N A to 75° for 3 min. Place the tube on ice and add the following reagents: 2/.d of primer ON1914 (25/zM), 4/xl of MgC12 (25 raM), 2/zl of 10× RT buffer (100 m M Tris-HC1, pH 8.8, 500 mM KCI, 1% Triton X-100), 2/xl of 10 m M each dNTP, 0.5/.d (20 units) of recombinant RNasin (Promega), and 1/zl (20 units) of avian myeloblastosis virus reverse transcriptase (Promega). Incubate at 42 ° for 40 rain. Precipitate the cDNA with ethanol as described earlier and resuspend the pellet in 11/xl of water. Prepare 10 PCR reactions containing 20 mM Tris-HC1 (pH 8.4), 50 mM KCI, 1 mM MgCl2, 0.5 /.~M each of ON2856 and ON1230, 10% glycerol, 0.2 mM each dNTP, and 1/zl of cDNA in a final volume of 100 /M. Cover the tubes with mineral oil and heat them for 2 min at 94°. Lower the temperature to 72 ° and add 0.5/zl (2.5 units) of Taq D N A polymerase for a "hot start." Incubate for 30 cycles at 94 ° for 30 sec, 50° for 30 sec, and 72 ° for 45 sec. Combine the PCR reactions and precipitate the amplified template with ethanol. Resuspend the D N A in 25 /zl of water and purify it by electrophoresis on a 3% NuSieve GTG agarose gel. Excise the 472-bp template band and recover the D N A using the Wizard system (Promega) as described previously for constructing the D N A library. The amplified template band is slightly smaller than the starting library because the primer ON1230 hybridizes 37 bp upstream from the 3' BstXI site of the spacer fragment.
Cloning and Sequencing of Enriched Sequences and Determining Peptide Binding Specificities To identify individual sequences that have been enriched during selection, it is necessary to clone and sequence the pools of amplified D N A
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DNA Pool M A S ( X )
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FI~. 3. Cloning vectors for sequencing and ELISA of enriched sequences. The partial nucleotide and predicted amino acid sequences of the amplifiedDNA library pool are shown on the top. The partial sequences for the plII phagemid vector pAFF6 (the arrow indicates the processing site for signal peptidase), MBP vector pLM190, and the polysome expression plasmid pLM193 are shown below. The NheI and KpnI cloning sites are underlined.
after each round. We have constructed cloning vectors that express the enriched peptide sequences either as N-terminal fusions to the phage coat protein pIII or as C-terminal fusions to the maltose-binding protein (MBP) (Fig. 3). The fusions are tested for binding activity using an ELISA, and the peptide-coding regions of the positive clones are sequenced. The phage E L I S A is also a convenient method for determining enrichment during polysome screening. Pools of sequences from each round of polysome screening are cloned into the plII phagemid vector, pAFF6, and a phage supernatant is prepared from the pool of transformants. If the phage pool is positive in the E L I S A , then individual clones from the same transformation mixture are tested for binding activity and sequenced. The MBP vector, pLM190, is used for expressing the peptides as Cterminal fusions to a cytoplasmic form of MBP (Fig. 3). Switching the flee end to C-terminal display can potentially affect binding activity, but there are several reasons for using an MBP ELISA. First, the MBP E L I S A is more affinity sensitive than the phage E L I S A , permitting a ranking of related sequences according to their affinities. 17 Second, for more detailed studies such as competition-binding assays, which require purified fusion protein, MBP can be expressed in large amounts as a soluble protein and is easily purifiedJ 8 iv C. M. Gates, E. L, Martin, and P. J. Schatz, personal communication (1995). 18p. j. Schatz, M. G. Cull, E. L. Martin, and C. M. Gates, Methods Enzymol. 267, Chap. 10, 1996 (this volume).
204
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Expressing a peptide fused to plII or MBP in E. coli is convenient for determining its binding specificity by ELISA, but may introduce biological biases which mask the binding activity that was selected for in vitro. For this reason, we have also developed an in vitro polysome-binding assay. 11 In this assay, a D N A template encoding the sequence to be tested is incubated in an in vitro reaction that is supplemented with radiolabeled UTP to label the newly synthesized mRNA. Polysomes, radiolabeled by virtue of the bound mRNA, are isolated and added to the immobilized target. After binding and washing, E D T A is added to dissociate the bound complexes and the radiolabeled m R N A is recovered and counted. In general, for a new target, we prefer the convenience of the phage system for sequencing and ELISA. If the sequence analysis reveals a strong consensus sequence that is negative in both the phage and MBP ELISA, then the polysome-binding assay is a useful alternative for determining the specificity of binding.
Procedure for Phage ELISA and Sequencing Digest 500 ng of the amplified D N A with NheI and KpnI. Extract once with phenol and three times with ether to remove any residual phenol. We avoid precipitating the digested library with ethanol because the random peptide-coding fragments are small (less than 60 bp) and do not precipitate efficiently. To ligate the fragments to pAFF6, combine 250 ng of the digested library with ligase buffer, 400 units of ligase, and 50 ng of vector that has been cut with NheI/KpnI and gel purified by the Geneclean method (Bio 101). Incubate overnight at 14°. Precipitate the ligated mixture with ethanol in the presence of glycogen as described earlier. Wash the pellet with 70% ethanol and resuspend it in 10/zl of water. Remove 5/zl and transform 20 /zl of E. coli strain XL1-Blue (Stratagene, San Diego, CA) by electroporation using SOC medium for the outgrowth step. 19 Grow the transformants for 1 hr at 37 ° without shaking, and plate 0.1 ml of undiluted to 10 -2 dilutions on LB-ampicillin (100/zg/ml) plates. Colonies from these plates are used to sequence and determine the binding specificity of the individual clones. To prepare a phage supernatant of the transformed pool, add 0.5 ml of the outgrown cells to 10 ml of phagemid growth medium (LB containing 0.25% K H 2 P O 4 , 0 . 1 % each of MgSO4 and glucose, and 100/.~g/ml of ampicilfin) and incubate overnight at 37°. Add 0.5 ml of the overnight culture to 10 ml of fresh phagemid growth medium and grow until the A600 reaches 0.6 to 0.8. Remove 1 ml of cells and infect with 5 x 10 9 plaque-forming units of VCSM13 helper phage (Stratagene) per ml of cells. Incubate at 19W. J. Dower, J. F. Miller, and C. W. Ragsdale, Nucleic Acids Res. 16, 6127 (1988).
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37° for 30 min without shaking to allow for attachment of the helper phage. Remove 0.4 ml and add to 2 ml of phagemid growth medium supplemented with 24 tzg/ml kanamycin and 0.024% (w/v) arabinose. Grow overnight at 37° with vigorous shaking and centrifuge the cultures at 5000 g for 10 min. Store the phage supernatants at 4° for 1 to 2 days or at - 2 0 ° for longer periods. To prepare a phage supernatant of individual clones, inoculate each colony in 5 ml of phagemid growth medium. Grow at 37 ° for 4 hr with vigorous shaking. Infect with VCSM13 helper phage as described earlier and add kanamycin and arabinose to a final concentration of 20/zg/ml and 0.02%, respectively. Grow overnight at 37°, centrifuge the culture, and store the phage supernatant at 4°. For the phage ELISA, prepare a polystyrene 96-well microtiter plate (Immulon 4, Dynatech). Each phage supernatant requires four wells: two are coated with the target and two are left as blanks. The concentration of target added to the wells must be optimized on a case-by-case basis. For a MAb, add 1 to 5/xg in 100/xl of PBS to each well. Incubate the plate at 37 ° for 1 hr. Wash the wells five times with PBS and block each well with 250 tzl of PBS/I% BSA. Incubate the plate at 37° for 1 hr and wash again five times with PBS. To each well, add 50/zl of PBS/0.1% BSA and 50/~1 of the phage supernatant. Incubate for 2 hr at 4°, shaking gently on a microtiter plate shaker. Wash the wells six times with PBS and add 50 t~l of horseradish peroxidase-conjugated to sheep anti-M13 IgG (Pharmacia) that has been diluted 1 : 5000 in PBS/1% BSA. Incubate for 1 hr at 4 ° on the plate shaker and wash the wells six times with PBS. Binding is detected by adding 100/xl of substrate [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)diammonium (0.2 mg/ml), 50 mM citric acid, pH 4/0.05% (v/v) hydrogen peroxide] and measuring the A405. Phage binding is scored as positive if the average A405 for the duplicate wells coated with the MAb is at least twofold greater than that of the corresponding control wells. To sequence the phage clones, prepare phagemid D N A from the phage supernatants using the Prep-A-Gene system (Bio-Rad). The primer ON-3 (5' CGATCTAAAGTTTTGTCGTCT 3') is a reverse sequencing primer that hybridizes 80 bp downstream from the KpnI site of pAFF6.
Procedure for MBP ELISA To clone into the MBP vector pLM190, add 50 ng of the cut and gelpurified vector to 250 ng of the NheI/KpnI cut amplified D N A pool. Ligate as described earlier and transform E. coli strain ARI 814 ~s by electroporation to isolate individual ampicillin-resistant colonies. Follow the steps for the MBP ELISA. is
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[ 1 11
Procedure for Polysome Binding Assay Ligate an individual sequence or a pool of amplified D N A sequences into the NheI/KpnI sites of pLM193. Electroporate E. coli strain XL1Blue and isolate individual ampicillin-resistant colonies as described earlier. Inoculate each individual colony into 10 ml of LB-ampicillin (amp), grow overnight at 37 °, and isolate plasmid D N A using the Qiagen plasmid purification system (Qiagen Corp., Chatsworth, CA). Linearize the plasmid by cutting 5 tzg with PacI, which cleaves just downstream of the 3' end of the spacer sequence. Extract the cut plasmid with phenol and precipitate it with ethanol. Resuspend the pellet in nuclease-free water to a concentration of about 250 ng//xl. Add 1/xg of the PacI-cleaved plasmid D N A encoding each sequence to be tested and a negative control, nonbinding, sequence to separate in vitro reactions supplemented with 10/zCi [ot-33p]UTP(Amersham, Arlington Heights, IL, 3000 Ci/mmol). Incubate the reactions and isolate the polysomes as described previously for polysome screening. Remove two 1-/M aliquots of the cleared polysome solution and precipitate each in 1 ml of trichloroacetic acid (TCA) that contains 25/xg of acetylated BSA. Collect the TCA precipitates on GF/C glass fiber filter disks (Whatman, Clifton, NJ) and count them in a liquid scintillation counter. Determine the average counts per minute (cpm) for each set of duplicates and use this value to normalize the polysome input for each sequence. Add approximately 100,000 cpm of the polysome preparation to the binding reaction and elute the m R N A as described previously. Precipitate, using TCA, two 40-/zl aliquots of the eluted m R N A and determine the average cpm that was recovered. If binding is specific, the cpm corresponding to the test sequence should be at least threefold greater than that obtained for the negative control sequence. Alternatively, the negative control can be the same polysome preparation added to beads preblocked with a competing ligand. Concluding Remarks The polysome display system is a recent biological method for screening peptide libraries. Unlike the cell-based methods, polysome screening relies on in vitro synthesis and amplification of the peptide sequences during each round of screening. This offers the potential of screening larger libraries and a more diverse collection of sequences since few biological biases should affect gene expression in vitro. It should also be possible to expand the structures of amino acids comprising the library by supplementing the in vitro system with suppressor tRNAs that have been chemically acylated with unnatural amino acids, a° 20C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, and P. G. Schultz,Science244, 182 (1989).
[11]
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A notable difference between phage display and the current polysome display system is the affinities of the recovered peptides. We find that highaffinity sequences (Kd values of less than 200 nM) predominate the pools of enriched sequences, which suggests that polysome display may be monovalent. This is in contrast to the multivalent phage and plasmid display systems which can recover ligands with affinities as low as 100/xM. 21Monovalent peptide display limits the range of ligand families that are recovered, but is very effective in selecting higher affinity sequences, especially if mutagenic PCR 22 is incorporated between rounds of selection. Repeated rounds of polysome screening result in certain sequences dominating the population, and often the most frequently occurring sequence will bind to the target with the highest affinity. For screening random libraries, it would be desirable to take advantage of the enormous diversity of sequences displayed on polysomes and enrich for lower affinity ligands. This could be accomplished by increasing the peptide valency by modifying the spacer sequence to increase the frequency of ribosome stalling. We estimate that an efficient stalling sequence at the 3' end of the spacer could result in up to five stalled ribosomes displaying the peptide. Such modifications of the spacer sequence should lead to further improvement and utility of the polysome display system.
21 E. M. Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor, and M. A. Gallop, J. Med. Chem. 37, 1385 (1994). 22 R. C. Cadwell and G. F. Joyce, PCR Methods Appl. 2, 28 (1992).