Generating molecular diversity by homologous recombination in Escherichia coli

Peter L. Wang1,2,3, Benny K.C. Lo4,5 and Greg Winter1,4

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We explored the use of recE-mediated homologous recombination to generate molecular diversity in Escherichia coli. Two homologous genes were placed on different phagemid vectors each comprising multiple EcoRI restriction sites and overlapping N- and C-terminal portions of ß-lactamase. By co-infection of these phage into RecE+ EcoRI+ E.coli, we were able to introduce double-strand breaks into these vectors, allowing efficient homologous recombination (in up to 10% of bacteria) by the recE pathway and selection of the recombinants by resistance to ampicillin. Recombination gave single crossovers; these were more frequent near the EcoRI sites and the recombination frequency increased with the target length and degree of homology. The system was used to create a large combinatorial chicken antibody library (1010) for display on filamentous phage and to isolate several antibody fragments with binding affinities in the 10–100 nM range.

Keywords: antibody phage display/chicken bursa/single-chain Fv library


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The creation and selection of molecular diversity have provided the most effective means of creating engineered proteins with desired binding and catalytic properties. This approach has led to the creation of human antibodies with therapeutic properties and also to enzymes with greatly enhanced catalytic rates. Diversity is readily created in vitro by a variety of strategies including error-prone PCR and DNA shuffling approaches (Hawkins et al., 1992Go; Stemmer, 1994Go). However, the size of molecular repertoires is limited by the efficiency of transformation of Escherichia coli by DNA ligated in vitro.

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., 1993Go). 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., 1994Go). 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, 2000Go); 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., 1989Go). 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., 1994Go). 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., 2002Go). Both RecET and {lambda} Red pathways have been exploited for recombinational cloning (Muyrers et al., 1999Go; Yu et al., 2000Go), including recent work using completely in vivo techniques (Li and Elledge, 2005Go). Homologous recombination is efficient in yeast and is commonly used for recombinational cloning (Ma et al., 1987Go) and occasionally for generating sequence diversity (Pompon and Nicolas, 1989Go; Cherry et al., 1999Go; Swers et al., 2004Go), 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., 1994Go). Gene conversion also contributes to antibody diversity in the rabbit (Becker and Knight, 1990Go) and probably in the cow, pig and horse (Butler, 1998Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Reagents

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 {lambda} Red genes under control of thermosensitive {lambda} 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., 1995Go).


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Table I. Strains and vectors from other sources

 
Recombination protocols

Phagemid particles were produced with KM13 or M13KO7 by standard protocols (Harrison et al., 1996Go); 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)Go: 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 {lambda} 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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Table II. Oligonucleotide primer sequences

 
Recombined scFv library

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-{alpha} (a gift from Anna Oates), LacZ (E.coli ß-galactosidase, Sigma), hen-egg lysozyme (Sigma); biotin–fluorescein was obtained from Molecular Probes. Standard phage display protocols were used (Harrison et al., 1996Go). 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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Early studies of filamentous phage (Ff) recombination took advantage of the fact that multiple phage can infect a single cell (Boon and Zinder, 1970Go). Similarly, two phagemid DNAs could be brought together simply by co-infection at high multiplicity. Co-infection has been utilized for Cre-lox recombination of single-chain Fv antibody fragments (scFv) and phage display (Sblattero and Bradbury, 2000Go).

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, ~10–4, 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., 1982Go). One group reported much higher recombination frequencies (2–9%) 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., 1994Go). 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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Fig. 1. Recombination vectors. (a) Co-infection recombination reporters: only crossover occurring between the frameshifts gives a functional TetR gene consisting of the segments marked by thick lines. (b) Double-strand break recombination reporters: vectors are shown after cleavage by EcoRI, irrelevant portions are shown with dashed lines; crossovers are shown in both homology arms, which will give rise to a circular product consisting of the segments marked by thick lines. (c) Phage display recombination vectors: these are based on the same recombination reaction as in (b); the resulting recombinant vector (pW324 as an example) can be used to express an scFv-gene III fusion for phage display. The pW324 diagram has been annotated in greater detail to illustrate the modular nature of the vectors.

 

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Table III. Wild-type recombination of phagemid vectors

 
Efficient recombination after double-strand breaks

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., 1985Go). Double-strand breaks can be produced in vivo by restriction endonucleases and one group used this with {lambda} phage infection to mimic the recombination process involved in intron homing (Eddy and Gold, 1992Go). 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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Table IV. Double-strand break recombination in WX13[pW43] and genotype dependence of double-strand break recombination

 
We examined the dependence of recombination on homology length, using a series of reporter vectors with different lengths of overlapping identical sequence (Figure 2a). Homology at both arms is required for recombination to occur, the minimum length of identity being between 11 and 30 bp. Recombination frequency increases with overlap length on either arm, but appears to plateau above 500 bp.



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Fig. 2. Effects of sequence length and similarity on recombination. (a) Double-strand break recombination of vectors with TetR and AmpR gene fragments of different overlapping lengths on the left and right crossover arms (as in Figure 1b). Ampicillin and tetracycline resistance frequencies gave similar results; plotted here is the mean of the two frequencies. (b) Double-strand break recombination of vectors (as in Figure 1c) with different scFv genes: cB1 and cL1 are chicken (Davies et al., 1995Go); B1.8 is mouse; scFv13 (Martineau et al., 1998Go), phOx15 (Marks et al., 1991Go) and phOx31E (Marks et al., 1992Go) are human. Each pair-wise combination is represented by one point. Recombination frequency = ampicillin-resistant colonies ÷ total colonies (growing in the absence of any antibiotic). (c) Pattern of recombination determined by DNA sequencing crosses between chicken scFv. The number of clones crossing over in each interval are shown above a diagram of a scFv with CDR regions indicated. See Supplementary material for sequences.

 
Recombination of non-identical sequences

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., 1987Go).

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, 1987Go); activity of the RecET pathway apparently further cripples the helper phage origin below a viable threshold. Indeed, the RecT and {lambda} beta proteins are known to bind to single-stranded DNA (Kolodner et al., 1994Go), an obligatory step in phage replication.



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Fig. 3. Interaction between Rec genes and helper phage. In both graphs, the percentage of antibiotic-resistant bacteria relative to total bacteria is plotted. (a) Helper phage (KanR) were infected into wild-type or RecET + bacteria (WX22 or WX13) carrying plasmids with or without a Ff phage origin (pUC119 or pUC19, both AmpR). (b) Acceptor and donor scFv phagemids were co-infected into bacteria that either had active RecET or the {lambda} Red genes carried by plasmid pW654. Recombinants are ampicillin-resistant and if infected with helper phage are also kanamycin resistant.

 
We therefore decided to place the recombination genes under inducible control, so that they could be turned off after recombination was completed to allow more efficient helper phage growth. We took advantage of a plasmid containing the {lambda} Red recombination genes under thermosensitive control; the {lambda} Red genes are very analogous to the RecET genes and perform equally well in recombinational cloning (Muyrers et al., 1999Go). Wild-type strains containing the {lambda} Red plasmid gave recombination frequencies (measured by ampicillin resistance) nearly as good as a RecE+ strain yet >100-fold more helper-infected recombinants (resistant to both kanamycin and ampicillin); still, this was only 20% of the total recombinants (Figure 3b).

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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Fig. 4. Recombination of chicken scFv repertoires. Two chicken bursa scFv libraries with SNPs engineered at three locations encoded the starting diversity. Partial fragments of these scFv were amplified and cloned into the acceptor and donor vectors to create the parental libraries. These were crossed to give the recombined library; DNA sequencing of random clones showed that all had one of the two SNP patterns expected of recombinants (frequencies given on the left). See Supplementary material for sequences.

 
For preliminary characterization of the functionality of the library, we undertook binding selections for five antigens (LacZ, TNF-{alpha}, ß-lactamase, lysozyme and fluorescein) using both the recombined and control libraries. Several binders with distinct sequences were identified for all antigens (Supplementary material), although for TNF-{alpha} the selections were dominated by a single clone for both libraries. Affinities for LacZ and TNF-{alpha} scFv clones were measured by surface plasmon resonance and the best affinity was 9.5 nM (Table V). Both TNF-{alpha} scFv inhibited the cytotoxic activity of TNF-{alpha} on mouse L929 cells (data not shown). Some of the affinities obtained were comparable to those from other large phage display libraries of fragments (Griffiths et al., 1994Go; Sblattero and Bradbury, 2000Go). However, we note similar affinities from both the recombined and control libraries (differing 140-fold in nominal size, 1 x 1010 vs 7 x 107 clones). We suggest that this may be due to the few samples analyzed, avidity effects during the phage selection due to multimerization of the scFv heads or the functional dominance by a limited portion of the sequence (e.g. heavy chain variable domain) in these chicken repertoires.


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Table V. Binding constants of selected scFv, determined by surface plasmon resonance

 
Other formats can be envisioned. The recombination system presented here uses two different vectors. A single vector system could use two types of restriction sites, with donor and acceptor species generated by differential DNA methylation on each of the two types of sites; such a system would allow multiple iterations of in vivo recombination without the need for re-cloning. Use of endonucleases that cut randomly (such as Type I restriction endonucleases or methylation-dependent endonucleases) would eliminate the need for specialized vectors altogether. Finally, since the recombination used here does not require any specific sequence (such as loxP), these methods can be used to create molecular diversity in proteins besides antibodies and for selection techniques besides phage display.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We thank Didrik Paus and Anna Oates for protein antigens and advice; John Young for help with chicken bursa cDNA; A.J.Clark, Amikam Cohen, Gordon Churchward, Svein Valla, Robert Caren and Chaitan Khosla for strains and plasmids; and A.J.Clark, Richard Kolodner and Susan Lovett for preliminary discussions on homologous recombination. P.L.W. was supported by a Hitchings-Elion fellowship from the Burroughs-Wellcome Fund and by the Medical Research Council. B.K.C.L. received a research fellowship from the Croucher Foundation, Hong Kong.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
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Received May 25, 2005; accepted May 26, 2005.





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