1Centre for Protein Engineering and 4Laboratory of Molecular Biology, University of Cambridge, Hills Road, Cambridge CB2 2QH, UK 2Present address: Department of Biochemistry, Stanford University Medical School, Stanford, CA 94305, USA 5Present address: St Edmund's College, University of Cambridge, Cambridge CB3 0BN, UK
3 To whom correspondence should be addressed. E-mail: plwang{at}stanford.edu
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Abstract |
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Keywords: antibody phage display/chicken bursa/single-chain Fv library
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Introduction |
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Hence there may be advantages to creating the diversity in vivo; for example, this can facilitate the construction of very large libraries by a process termed combinatorial infection (Waterhouse et al., 1993). A large combinatorial repertoire of antibody Fab fragments was created by Cre-lox mediated recombination, after infection of a phage-encoded light chain repertoire into bacteria containing a plasmid-encoded heavy chain repertoire (Griffiths et al., 1994
). In that case the diversity was reassorted by crossover at a single fixed site (loxP). By contrast, homologous recombination would reassort diversity by crossover at many sites (Wang, 2000
); this has the potential to generate wider diversity and does not require an extraneous sequence such as loxP to be present.
Cre-lox mediated recombination is highly efficient, but homologous recombination in wild-type strains of E.coli mediated by RecA is not, particularly for plasmid DNAs. Alternate pathways mediated by phage recombination proteins are more efficient, especially when the target DNA contains double-strand breaks (Luisi-DeLuca et al., 1989). One example is the RecE pathway, mediated by the recE and the recT genes of the rac prophage present in many strains of E.coli but not normally active. RecE is a 5' to 3' exonuclease while RecT protein can promote annealing of homologous DNA single strands (Kolodner et al., 1994
). RecET expression is activated by sbcA mutations and further enhanced by inactivation of the RecBCD exonuclease. A similar pathway is the Red pathway of phage lambda, mediated by the genes exo, bet and gam (Court et al., 2002
). Both RecET and
Red pathways have been exploited for recombinational cloning (Muyrers et al., 1999
; Yu et al., 2000
), including recent work using completely in vivo techniques (Li and Elledge, 2005
). Homologous recombination is efficient in yeast and is commonly used for recombinational cloning (Ma et al., 1987
) and occasionally for generating sequence diversity (Pompon and Nicolas, 1989
; Cherry et al., 1999
; Swers et al., 2004
), but yeast has been a less favored host for protein engineering than E.coli.
In this work, we explored the use of homologous recombination and combinatorial infection to generate large highly diverse antibody libraries. We decided to create the library from chickens as natural chicken antibodies are created by gene conversion, a homologous recombination process that copies bits of sequence from multiple pseudogenes into the single active antibody locus (Reynaud et al., 1994). Gene conversion also contributes to antibody diversity in the rabbit (Becker and Knight, 1990
) and probably in the cow, pig and horse (Butler, 1998
).
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Materials and methods |
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Antibiotics were used at the following final concentrations (µg/ml): ampicillin (Amp) 100, chloramphenicol (Cam) 30, tetracycline (Tet) 10, kanamycin (Kan) 25, gentamycin (Gm) 10, trimethoprim (Trm) 10, streptomycin (Str) 25 and spectinomycin (Spc) 50. When growing in liquid culture, only antibiotics for the plasmid markers were used (not for chromosomal or F-encoded markers). 2x TY is 16 g tryptone, 10 g yeast extract, 5 g NaCl per liter; 2x TYG is 2x TY + 1% glucose.
Strains and plasmids (Table I)
pW18 and pW19 were made by ligating a 1.3 kb AvaI (blunted) + EcoRI pBR322 fragment into HincII + EcoRI pBluescript II SK + or pBC-SK + (Stratagene) followed by SalI or NheI digestion, fill-in and re-ligation. pW43 was made by ligating a 3 kb fragment from pMB4 SalI + partial HindIII digest to SalI + HindIII pGB2. pW654 was made by ligating a 3.2 kb MspA1I + MfeI pZS20 fragment to SmaI + EcoRI pW653 and expresses Red genes under control of thermosensitive
repressor. pW653 contains an RK2 origin from pJB658, mouse dihydrofolate reductase gene for TrmR and the pBluescript II SK + polylinker; it was constructed using PCR products, as were the rest of the other vectors used. The sequences of pW256, pW260, pW357, pW364 and pW654 are provided in Supplementary material available at PEDS Online. The GmR gene of pW256 is from pBSL141 (Alexeyev et al., 1995
).
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Phagemid particles were produced with KM13 or M13KO7 by standard protocols (Harrison et al., 1996); when particles were not intended for phage display, media with 1% glucose was used.
For co-infection, phagemid particles for each vector at a multiplicity of infection (MOI) of 10 were added simultaneously to log-phase bacteria, incubated without shaking at 37°C for 1 h, then titered by spotting 10 µl of serial dilutions on to agar plates. Note that because of the high MOI, sufficient helper phage is already present at initial infection; no additional helper phage is needed (nor does it provide any benefit). When working with pW654, all cultures were grown at 30°C before phagemid infection.
Infection into vector-carrying bacteria was performed following Caren et al. (1994): phagemid particles for one vector were added at MOI 10 to stationary-phase bacteria carrying the other vector, incubated without shaking at 37°C for 1 h, then titered. Phagemid particles were produced with R408 rather than M13KO7 so that the vector KanR marker could be followed.
Library construction
Oligo-dT primed cDNA was made from the bursas of 6.5-week-old line 0 (L0) and Rhode Island Red (RIR) chickens with the kind help of John Young (Institute for Animal Health, Compton, UK). Primer sequences are given in Table II. For L0 cDNA, VH was amplified with HBaPel1/HFoLink1, then re-amplified with HBaPel2/phosphorylated HFoLink2; VL was amplified with LBaLink1/cCLFoNot, then re-amplified with phosphorylated LBaLink2/cCLFoNot. For RIR cDNA, the PCRs were similar except that HBaPel1Tag, HBaPel2Tag, HFoLink2Tag and cCLFoNotTag were used. VH and VL PCRs were treated with exonuclease and then joined by PCR assembly using HBaPel2/cCLFoNot or their Tag versions. The assembled scFv were digested with SfiI + NotI and ligated to SfiI + NotI pW260 (for L0) or pW256 (for RIR) to make the original scFv libraries. The control unrecombined library was made by amplifying scFv from the original libraries with A177seq9/pHEN-SEQ (L0) and pelBback/rec4seq1 (RIR) and ligating to SfiI + NotI pW324. The parental recombination libraries were made by amplifying fragments from the original libraries with cVHfr3BaSfi/pHEN-SEQ (L0) or pelBback/cVLfr3FoNot (RIR) and ligating to SfiI + NotI pW260 (L0) or pW256 (RIR).
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Six liters of 2x TYG + Trm + Spc were inoculated 1:50 with TG1[pW43][pW654] overnight culture and shaken at 30°C to OD = 2. The temperature was raised to 37°C by adding 70°C media. Phagemid particles from each parental recombination library were added at MOI 5 and the cultures were incubated without shaking at 37°C for 1 h; an aliquot was titered to determine recombination frequency. Cultures were centrifuged and resuspended in 4 l of 2x TY + Amp + 0.1% glucose, then shaken at 30°C. Saturated cultures were centrifuged and resuspended in 150 ml of 2x TYG + 20% glycerol and frozen. A 24 ml volume of glycerol stock was added to 7 l of 2x TYG + Amp and grown to saturation; plasmid DNA was prepared with the QIAGEN HiSpeed Midiprep kit. Plasmid DNA was electroporated into TG1tr and transformants grown on Amp + 1% glucose plates.
scFv phage selection and kinetic measurements
Protein antigens were chemically biotinylated: TEM-1 ß-lactamase (a gift from Didrik Paus), TNF- (a gift from Anna Oates), LacZ (E.coli ß-galactosidase, Sigma), hen-egg lysozyme (Sigma); biotinfluorescein was obtained from Molecular Probes. Standard phage display protocols were used (Harrison et al., 1996
). About 1012 phagemid particles were used in each selection round. First round phagemids were panned on antigens bound to Neutravidin microtiter wells (Pierce Chemical); second round phagemids were mixed with 10 nM antigen solutions followed by capture on streptavidin Dynabeads (Dynal); third round was similar but divided between 10 and 1 nM antigen solutions. Monoclonal scFv supernatants were screened by ELISA for binding to streptavidin-coated microtiter wells with or without antigen. For kinetic measurements, scFv was purified on NiNTA agarose (Qiagen), followed by gel filtration to obtain the monomer fraction just prior to use. About 100 RU of antigen was coupled to a streptavidin chip; three different scFv concentrations were injected in the manufacturer's HBS-EP buffer at 25°C on a BIACORE-2000 (Pharmacia). Triplicate sensorgrams were averaged after subtraction of a buffer injection sensorgram. Kinetic constants were calculated by global fitting using a 1:1 Langmuir model.
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Results and discussion |
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Recombination of infected vectors
To determine the frequency of recombination, we used the reporter system for testing co-infection and recombination pictured in Figure 1a. There are two phagemid vectors, each marked with a different antibiotic resistance (CamR or AmpR); they also carry TetR genes that are inactive owing to frameshift mutations at different sites. Recombination of the TetR genes between the frameshifts recreates an active gene. Most bacteria are infected by both vectors (that is, resistant to both ampicillin and chloramphenicol); however, the frequency of recombination events giving tetracycline-resistant bacteria is low, 104, in both wild-type and RecE+ strains (Table IIIa). This is about the same as the frequencies from studies using co-transformation (rather than co-infection) of reporters similar to ours (James et al., 1982
). One group reported much higher recombination frequencies (29%) using a somewhat different protocol but similar vectors: one vector was already resident in the bacteria and only the second vector was brought in by infection; the vectors used different antibiotic markers than ours (Caren et al., 1994
). We tested this protocol using both sets of vectors and still found low recombination frequencies (Table IIIb) similar to those obtained by co-infection. Such frequencies are too low to be useful for repertoire engineering, especially in the absence of any generic selection for recombinants.
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Experiments using DNA transformation suggested that DNAs with double-strand breaks could be very efficiently recombined in strains with an active RecE pathway (Symington et al., 1985). Double-strand breaks can be produced in vivo by restriction endonucleases and one group used this with
phage infection to mimic the recombination process involved in intron homing (Eddy and Gold, 1992
). We therefore designed vectors with appropriately located EcoRI recognition sites (Figure 1b). These vectors would be introduced by co-infection; after cell entry and conversion to double-stranded form, they would be cleaved by EcoRI endonuclease to give linear fragments for recombination. Recombination at two points is required to regenerate a circular vector. The recombinant vector should retain the antibiotic marker of one of the parental vectors, called the acceptor; the other parental vector is called the donor.
Initial experiments used vectors with incomplete fragments of antibiotic resistance genes (TetR and AmpR) at each crossover arm. In the absence of the partner carrying overlapping gene sequence, each vector was efficiently restricted by EcoRI (very low transduction to chloramphenicol or gentamycin resistance). For this work, we define recombination frequency as the fraction of all bacteria that show evidence of a recombination event; this is typically the ratio of the bacteria that acquire a specific antibiotic resistance divided by the total bacteria in the culture (colony titers on plates lacking any antibiotic). When both vectors are co-infected, the recombination frequency was high (30%, as measured by tetracycline or ampicillin resistance) and recombinants always carried the resistance marker of the acceptor but not the donor vector (Table IVa). For most of the remaining 70% of bacteria, restriction was efficient but recombination did not occur as they contain no plasmid at all (data not shown). Efficient double-strand break recombination depends mainly on activation of RecET (sbcA) but also on inactivation of RecBCD nuclease (recBC); specific disruption of RecE reduces recombination to background levels (Table IVb).
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To generate new sequence diversity one needs to recombine non-identical sequences. The recombination vectors were configured to accept scFv inserts and to generate products suitable for phage display (Figure 1c); recombinants will be ampicillin resistant. We created vectors carrying scFv from different species (chicken, mouse, human) and crossed them in all combinations. Recombination frequency increases with sequence identity, apparently following a power law (Figure 2b).
Recombinant plasmids had the expected overall structure based on restriction enzyme and PCR analysis (data not shown). Recombinants from crosses between chicken scFv genes were sequenced. All had a single crossover point where sequence switched from that of one scFv to the other; as expected, the acceptor scFv sequence was 5'. There were no point mutations, indicating that in vivo recombination is a high-fidelity process. Crossovers were distributed throughout the scFv gene (Figure 2c) but were clearly biased towards the 5' and 3' ends of the gene, the double-strand break sites. Such bias has been observed in other double-strand break recombination models (Abastado et al., 1987).
Recombination genes have deleterious effects on helper phage
Production of phage display particles requires superinfection with a helper phage, since phagemid vectors such as those used here lack most of the genes necessary for the viral life cycle. Commonly used helper phage such as M13KO7 and KM13 encode kanamycin resistance allowing infection with helper phage to be selected for. We were surprised to find that only a very small fraction of bacteria from in vivo recombination cultures could be infected by helper phage to become kanamycin resistant (data not shown). This helper infection resistance was not due to the recombination event per se, or to in vivo restriction, or to our particular vectors; the same effect was observed in a much simpler system, helper infection of RecE+ bacteria carrying a plasmid with a filamentous phage origin (Figure 3a). If either the bacteria were wild-type for recombination or the plasmid did not contain a phage origin, helper infection resistance was not seen. Helper phage replication origins are partially crippled by design, to minimize interference with the phage origin on the plasmid (Vieira and Messing, 1987); activity of the RecET pathway apparently further cripples the helper phage origin below a viable threshold. Indeed, the RecT and
beta proteins are known to bind to single-stranded DNA (Kolodner et al., 1994
), an obligatory step in phage replication.
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Functional repertoire from in vivo recombination
In order to demonstrate that in vivo recombination could generate useful molecular diversity, we created and selected a large phage display library of chicken scFv fragments. Sequences were derived from two original libraries of chicken scFv, amplified from bursa and engineered with single-nucleotide polymorphisms (SNPs) at the 5' end, middle and 3' end. As a control unrecombined library, these original scFv (7 x 107 clones) were amplified and cloned directly into a phage display vector. Parental recombination libraries were constructed by amplifying fragments of the original scFv and cloning into acceptor and donor vectors (Figure 4). These parental libraries were packaged as phage and co-infected into a recombination host strain, resulting in 1.4 x 1010 recombinants. To maximize phage production and provide a large permanent stock of the recombined library, plasmid DNA from the recombinant cells was transformed into the wild-type strain TG1 to give 1 x 1010 colonies. Sequencing of recombined library clones showed that the expected recombination had occurred in all cases and crossovers were distributed relatively proportionately in the two intervals distinguished by the SNP patterns (Figure 4). Our results show that we can successfully use homologous recombination together with combinatorial infection to generate large genetically diverse antibody libraries.
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Acknowledgements |
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Received May 25, 2005; accepted May 26, 2005.
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