Bacteriophage a (Phage Display/Biopanning/D Protein/Site-Specific Recombination/Libraries) NAT STERNBERG and RONALD H
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Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1609-1613, February 1995 Genetics Display of peptides and proteins on the surface of bacteriophage A (phage display/biopanning/D protein/site-specific recombination/libraries) NAT STERNBERG AND RONALD H. HoEss Dupont-Merck Pharmaceutical Company, Experimental Station E328/B33, Wilmington, DE 19880-0328 Communicated by Sankar Adhya, National Cancer Institute, Bethesda, MD, October 14, 1994 (received for review August 12, 1994) ABSTRACT The display of peptides or proteins on the age processing has also shown that D protein is assembled as surface of viruses is an important technology for studying trimers, which appear as prominent protrusions on the capsid peptides or proteins and their interaction with other mole- surface (8). This feature suggests that peptides or proteins cules. Here we describe a display vehicle based on bacterio- fused to D protein are likely to be accessible for binding to phage A that incorporates a number of features distinct from target molecules. Recently, a display system using the V tail other currently used display systems. Fusions of peptides or protein of bacteriophage A has been reported (9). protein domains have been made to the amino terminus ofthe 11-kDa D protein ofthe A capsid. These fusions assemble onto the viral capsid and appear to be accessible to ligand inter- MATERIALS AND METHODS actions, based on the ability of a monoclonal antibody to Construction and Use of Bacterial Strains and Phage*. recognize an epitope fused to the D protein on phage heads. Escherichia coli strain YMC supF was used for titering plaque- To produce large D fusion display libraries and yet avoid the forming units (pfu). E. coli NM522 F' lacIqA(lacZ) M15 cumbersome task of cloning many fragments into A DNA, we proA+B+/supE thiA(lac-proAB) containing D fusion plasmids have used the Cre-loxP site-specific recombination system in was used as a source of extracts containing D fusion protein. vivo to incorporate plasmids encoding the D fusions into the Strains NS2973 and NS2974 are JM109 recAI supE44 lysogenic phage genome. Finally, we show that D fusion proteins can be for Aimm434 ninS and Aimm434 ninS Cre+, respectively. added in vitro to phage lacking D protein and be assembled NS3762 is E. coli strain N99 sup°recAS6 tetr(ADamlS b538 onto the viral capsid. cIts857 ninS). It was generated by infecting strain N99 recA+ with ADamlS b538 cIts857 ninS and selecting A lysogens at Surface display of peptides or proteins has become a powerful 32°C as described (10). Since the b538 deletion in this phage tool in protein engineering and for discovering ligands that removes the A integration system, the frequency of lysogeny by bind receptors. The features that make this technology valu- this phage is low, and the phage presumably insert into the able are (i) the ability to create large molecular libraries of bacterial chromosome by illegitimate recombination. A lyso- variants and (ii) in vitro selection techniques that allow effi- gen containing tandemly integrated prophage was selected by cient searches of the library without testing each member virtue of its ability to produce 10-100 phage per cell when the individually. Smith and coworkers (1, 2) first demonstrated lysogen was thermally induced (11). It should be noted that the this technology by using phage fd where amino-terminal fu- yield of A phage in this experiment is high despite the lack of sions were made to the minor coat protein gIII. Since that time the normally essential phage D protein because the phage considerable progress has been made in constructing large contains a net 21.5% which obviates the libraries of random peptides, and a variety of proteins includ- genome deletion, ing antibodies have been displayed (for recent review, see ref. need for D. Finally, the tandem lysogen was made recA- by 3). A number of other display systems have been developed P1-mediated transduction of the recA56 allele from strain that use a bacterial surface rather than the viral capsid as the NS1426 srl::TnlO recAS6. To produce phages from NS3762, an display surface (4). A feature common to both phage and exponentially growing culture was induced by shifting the cells bacterial systems is that the displayed molecule(s) must be from 32°C to 42°C for 15 min to inactivate the cIts857repressor secreted across the bacterial membrane. We describe here a and then shifted to 38°C for 40 min. The culture was com- system where secretion is not required for phage display. pletely lysed by 45 min after the temperature shift and pro- Bacteriophage A is a virus that, in its lytic mode, assembles duced 10-50 phage per cell. intracellularly prior to release of viral particles from the cell. To construct a A D- phage containing a loxP site, ADamlS Much is known about the structural proteins that make up the imm2l nin5 was grown in a dam- host, and the phage DNAwas viral shell and about the pathway(s) by which these proteins cleaved at the unique Xba I site at map coordinate 24,508. The assemble (5). The 11-kDa D protein encoded by the phage resulting two A arms were ligated to an annealed complemen- appears to play a role in stabilizing the phage prohead as it fills tary set of oligonucleotides, 5'-AGCTTATAACTTCGTAT- with DNA (6, 7). Normally D protein is essential for phage AATGTATGCTATACGAAGTTATGTTTAAAC-3' and head morphogenesis. However, when the size of the phage 5' -AGCTGTTTAAACATAACTTCGTATAG- genome is <82% of wild type, phage can be assembled in the CATACATTATACGAAGTTATA-3', that contain the loxP absence of D protein and are stable in the presence of 10 mM sequence and are flanked by 4 bp of single-stranded Xba I Mg2+, although they are exquisitely sensitive to disruption by ends. The ligated DNA was packaged into A phage using a EDTA (6). The conditional requirement for D protein in viral Gigapack II system (Stratagene). Lysates were made from assembly suggests that this protein may be the ideal choice for individual plaques, and these were tested for the presence of fusion with peptides or protein domains for display on the viral capsid. Recent work using cryo-electron microscopy and im- Abbreviations: Kmr, kanamycin resistant (resistance); Kmsi, kanamy- cin sensitive; Ap, ampicillin; Apr, Ap resistant (resistance); pfu, plaque-forming unit(s); AII, angiotensin II; Bi, Bi domain of protein The publication costs of this article were defrayed in part by page charge G from group G Streptococcus; IPTG, isopropyl f3-D-thiogalactoside. payment. This article must therefore be hereby marked "advertisement" in *The various strains used in this work can be obtained from the accordance with 18 U.S.C. §1734 solely to indicate this fact. authors. 1609 Downloaded by guest on September 25, 2021 1610 Genetics: Sternberg and Hoess Proc. Natl. Acad Sci. USA 92 (1995) the loxP site by PCR amplification of 1 Al of lysate by using two EDTA sensitivity of phage was tested. Phage were diluted into primers, 5 '-GGAATTGGTTAGCAAGTTACTACCG-3' either 10 mM Tris, pH 7.5/10 mM MgCl2 (TM) or 10 mM Tris, and 5'-ATTGAAGCAAATCTGAAACCTATTA-3', that pH 7.5/10 mM EDTA (TE) and incubated for 10 min at 37°C. flank the A Xba I site. Serial dilutions were then titered on YMC cells. Construction of D Fusion Vectors. The A D gene was am- Generation of A, D-Fusion Plasmid Cointegrates by Cre- plified by PCR using primers, 5'-GTAAGCCATGGTTAT- loxP Recombination. Strains NS2973 and NS2974 containing GACGAGCAAAG-3' and 5 '-GTTCGAATTCCTATTA- D fusion plasmids with loxP sites were grown to an OD600 of AACGATGCTGATTGCC-3', that contain the restriction -0.2 in Luria-Bertani (LB) medium containing ampicillin sites Nco I and EcoRI (underlined), respectively. After cleav- (Ap) at 100 ,ug/ml at 37°C. One milliliter of cells was then age by Nco I and EcoRI, the DNA was cloned into the Nco I centrifuged, and the pellet was resuspended in 100 ,ul of a A and EcoRI sites of pTrcHisA (Invitrogen), resulting in plasmid D-loxP lysate at a multiplicity of infection of 1. After absorp- pNS3785. This places the wild-type D gene under the control tion at 37°C for 10 min, infected cells were diluted to 1 ml in of the pTrc promoter. To create peptide or protein fusions to LB medium containing Ap (100 ,tg/ml), 10 mM MgCl2, and 1 D protein, the vector was modified by cloning an oligonucle- mM isopropyl f3-D-thiogalactoside (IPTG). Cultures were otide adaptor, 5'-CATGTCTGACCGTGTTTACATCCAC- grown for -2 hr at 37°C, and then cells were lysed. A drop of CCGTTCGGCGCGCCATCTGTTAGCGGCCGCAC-3', chloroform was added, and cell debris was removed by cen- into the unique Nco I site at the amino terminus of the D trifugation. This lysate was used to infect an exponential coding sequence to create pRH800. This oligonucleotide en- culture of YMC at a multiplicity of infection of 1, and Ap- codes the eight amino acids of the peptide hormone angio- resistant (Apr) transductants containing the desired A-plasmid tensin II (AII) in frame with D followed by unique Asc I and cointegrates were selected (17). Not I restriction sites (underlined), respectively. DNA encod- Preparation of D Fusion Extracts. Cultures (20 ml) of ing the Bi domain of protein G from group G Streptococcus NM522 containing either plasmid pNS3785, pRH804, or (Bi) was PCR amplified from pET-16BStrpG (12) using prim- pRH815 were grown at 37°C to OD600 of -0.25. IPTG was ers 5'-GTGATCGGCGCGCCAGACATCTTGGCTGC- then added to 1 mM, and the cultures were grown for an TCTGCCG-3' (forward) and 5'-TAGATCCATGCGGC- additional 2 hr at 37°C.