Appl Microbiol Biotechnol (2008) 80:447–458 DOI 10.1007/s00253-008-1576-1

APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY

Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display Jonas Kügler & Simone Nieswandt & Gerald F. Gerlach & Jochen Meens & Thomas Schirrmann & Michael Hust

Received: 9 May 2008 / Revised: 11 June 2008 / Accepted: 11 June 2008 / Published online: 18 July 2008 # Springer-Verlag 2008

Abstract The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen–host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of J. Kügler : S. Nieswandt : T. Schirrmann : M. Hust (*) Abteilung Biotechnologie, Institut für Biochemie und Biotechnologie, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany e-mail: [email protected] G. F. Gerlach : J. Meens Institut für Mikrobiologie, Stiftung Tierärztliche Hochschule Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany Present address: J. Kügler : S. Nieswandt Helmholtz Zentrum für Infektionsforschung, Inhoffenstr. 7, 38124 Braunschweig, Germany

which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species. Keywords Phage display . Mycoplasma . Screening methods . Open reading frame enrichment

Introduction The identification of immunogenic proteins of pathogens is very important for the development of diagnostic tools and vaccines (Maas et al. 2006; Hoelzle et al. 2007). A common method for the identification of immunogenic proteins is a two-dimensional polyacrylamide gel electrophoresis (PAGE) of cultured bacterial pathogens, immunoblot using sera of infected patients or animals followed by mass spectrometry or microsequencing (Meens et al. 2006; Huntley et al. 2007; Delvecchio et al. 2006; Sellman et al. 2005; Jacobsen et al. 2005). This method is limited, because differentially expressed proteins, e.g. dependent on pathogen–host interaction, are not detected. Therefore, the identification of the complete immunoproteome requires a recombinant deoxyribonucleic acid (DNA) approach whereby immunogenic polypeptides can be identified independent of their in vitro expression. When working with Mycoplasma species, the results achieved by using common Escherichia coli expression libraries are limited due to their uncommon usage of the opal stop codon (UGA) to encode tryptophan. The use of polypeptide display libraries generated from genomic fragments of pathogens could overcome this limit-

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ation by combining the expression of a large number of small unknown antigen fragments with a convalescent serum-based selection process termed “panning.” The phage display selection strategy bases on the physical linkage of genotype and phenotype by fusing short gene fragments to the minor coat protein III gene (gIII) of the filamentous bacteriophage M13 resulting in the expression of the encoded fusion protein on the phage surface and thereby allowing affinity purification of the gene of interest by the binding function of the corresponding polypeptide. Mainly, the genes encoding the polypeptide::pIII fusion proteins are on a separate plasmid, termed phagemid (Breitling et al. 1991; McCafferty et al. 1990; Smith 1985). Besides M13, the lytic E. coli phage T7 (Sidhu and Coide 2007), limited to peptide display and short complementary DNA (cDNA) libraries, and the lytic phage Lambda (Beghetto et al. 2006) are used, whereas the nonlytic phage M13 is the most-used phage display system, especially for antibody selection (Hust and Dübel 2004; Taussig et al. 2007; Konthur et al. 2005). To date, cDNA or genomic M13 phage display libraries are used for the identification of protein–ligand interactions (Cochrane et al. 2000), protein–protein interactions (Hertveldt et al. 2003) selection of lectins (Yang et al. 2007), identification of allergenic proteins (Rhyner et al. 2004; Kodzius et al. 2003; Crameri et al. 2001) or the profiling of multiple sclerosis-associated autoantibodies (Govarts et al. 2007) These methods, however, have the disadvantage that phage-based polypeptide expression requires the in-frame insertion between the signal sequence and the gIII of the M13 phage to obtain expression. According to the calculated rate of non-directional cloning, only 1 of 18 (5.55%) cloned DNA fragments result in open reading frames (ORFs). Furthermore, stop codons in the gene fragments can abrogate the translation of the gene::gIII fusion required for the enrichment. Due to the selection pressure, phage display vectors without or with out-of-frame inserts are more efficiently propagated than vectors containing a continuous ORF insert, leading to an increase in junk clones during the panning. Therefore, it would be instrumental to enrich ORFs before selection. This was first achieved by cloning gene fragments in fusion with resistance marker genes to promote the enrichment of gene fragments which are in-frame with the selection marker (Faix et al. 2004). The disadvantage of this method is the need to remove the resistance marker gene after ORF enrichment and the loss of library complexity due to this second cloning step. This removal can be done by subcloning of the ORF fragment (Faix et al. 2004) or by constructing a resistance gene flanked by loxP sites which could be removed in vivo by Cre recombinase (Zacchi et al. 2003) In a prior work, we developed a system allowing the combined enrichment and display of ORFs without any subcloning steps. The proof of principle of this concept was demonstrated by cloning DNA fragments encoding two different Salmonella Typhimurium oligopeptides in three dif-

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ferent reading frames into the phage display vector pHORF2 and packaged using the Hyperphage (Rondot et al. 2001; Soltes et al. 2007; Hust et al. 2006). This helperphage has a truncated gIII on the phage genome, and the only source of pIII—an essential M13 coat protein—is the polypeptide::pIII fusion protein encoded on the phagemid. Infective phage particles can only be produced if cloned DNA inserts are in frame with the pelB leader sequence and gIII. The produced phage particles, containing an ORF and displaying the corresponding polypeptide, can be used directly for phage display. Mycoplasmas belong to the class Mollicutes and are distinguished from other bacteria by their total lack of a cell wall. They have the smallest genome (580–1,358 kb) of all self-replicating bacteria, the GC content of the genome is very low (24–40%), and they use the opal stop codon UGA to encode tryptophan (Sirand-Pugnet et al. 2007a, b). Due to the small genome size, Mycoplasma genitalium is used as starting point for the construction of artificial genomes (Gibson et al. 2008) and for analysis of minimal genome settings (Glass et al. 2006). Beyond this, Mycoplasma was used for the first genome transplantation (Lartigue et al. 2007) and the first demonstration of the 454 Life Sciences parallel pyrosequencing technology (Margulies et al. 2005). In general, Mycoplasma species are strictly host specific, and many of them are pathogenic for their respective host. In pigs, for example, M. hyopneumoniae is the cause for enzootic pneumonia (Meens et al. 2006; Madsen et al. 2008) a disease responsible for high economic losses. Diagnosis of the pathogen is difficult, and current vaccines are expensive to produce as they are based on whole cells which have to be cultured in media containing 20% of porcine serum. For the development of improved diagnostic tools and vaccines, immunogenic polypeptides have to be identified. To date, this knowledge is limited (Hsu et al. 1997; Strasser et al. 1991; Meens et al. 2006; Kim et al. 1990; Futo et al. 1995) as the identification of immunogenic polypeptides by recombinant DNA technology was hampered by the usage of UGA encoding tryptophan and the low GC content. In the work presented here, we applied the Hyperphage technology to construct a M. hyopneumoniae genomic phage display library in order to identify phage-encoding immunogenic polypeptides of M. hyopneumoniae.

Materials and methods Construction of pHORF3, pHORF3-Mhp651 and cloning of the identified immunogenic polypeptides E. coli culture and standard cloning procedures were performed according to Sambrook and Russell (2001). The phage vector pHORF3 (Fig. 1) was constructed from pHORF2 (Hust et al. 2006) by using the oligonucleotide

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Fig. 1 Schematic overview about library construction, Hyperphage-based ORF enrichment, and the panning procedure. Chromosomal DNA was sheered by sonification. The genomic DNA fragments were polished and cloned into the phagemid pHORF3. The initial genomic library was packaged using Hyperphage to enrich continuous ORFs in library. The phage library was used for panning on convalescent pig serum. Binders were selected by panning and further analysed. Lac Pr. Promoter of the bacterial lac operon, RBS ribosome binding site, pelB sequence encoding the signal peptide of bacterial pectate lyase, mediating protein secretion into the periplasmic space, ochre ochre stop codon; amber amber stop codon, tag hexahistidine and c-myc tags, terminator sequence terminating transcription, bla β-lactamase gene for ampicillin resistance, ColE1 bacterial origin of DNA replication, M13 ori intergenic region of phage f1

primers to replace both NotI restriction sites by a PmeI restriction site. The DNA sequence encoding the section 55–1,442 bp of Mhp651 of M. hyopneumoniae strain 232 (Meens et

al. 2006) was amplified by polymerase chain reaction (PCR) using the Phusion polymerase (NEB, Frankfurt am Main, Germany) and cloned into the PmeI site of pHORF3.

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Transformations of E. coli XL1-Blue MRF′ (Stratagene, Amsterdam, The Netherlands) with the constructs were done by electroporation according to the manufacturer’s instructions. Construction of a M. hyopneumoniae genomic phage display library A schematic overview of the construction, ORF selection and selection of immunogenic polypeptide displaying phage particles by panning is given in Fig. 1. The genomic DNA of M. hyopneumoniae strain J was isolated as previously described (Meens et al. 2006) Briefly, 60 μg genomic DNA in 300 μL dH2O was sonicated (six to nine times, 2 min, with 50% intensity and five cycles using Sonotrode MS73, Bandelin, Berlin, Germany). The fragmented DNA was precipitated by adding 30 μL 3 M sodium acetate (pH 5.2) and 750 μL ethanol for 2 min at room temperature (RT) and 5 min centrifugation with 16,000×g. The pellet was washed two times with 70% ethanol, dried and dissolved in 35 μL dH2O. Afterwards, the DNA was separated by 1.5% agarose gel electrophoresis. DNA fragments with a size of 200–1,200 bp were isolated using the GFX kit (GFX PCR DNA and gel band purification kit, GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. DNA fragments were polished for 10 min at 25°C in 50 μL reaction volume using 5 μg of sonicated genomic DNA, deoxyribonucleotide triphosphate (dNTP) mix (1 mM of each dNTP), New England Biolabs (NEB) buffer 2, 15 U T4 DNA polymerase (NEB, Frankfurt) and 5 μg bovine serum albumin (BSA). Afterwards, 12.5 U E. coli large Klenow fragment (NEB) were added and incubated for 10 min at 25°C followed by another incubation for 16 h at 16°C. The DNA was purified using the GFX kit (GE Healthcare). In parallel, 3 μg vector pHORF3 was digested using NEB buffer 4, BSA and 30 U PmeI (NEB) at 37°C overnight in a 60 μL reaction volume. The digestion was heat-inactivated at 65°C for 10 min. The dephosphorylation of the digested vector was performed by addition of 0.5 U calf intestine phosphatase (NEB) and incubation for 30 min at 37°C. This step was repeated before the reaction was stopped, and the vector fragment was purified using the GFX kit (GE Healthcare). One hundred nanograms of the digested and dephosphorylated vector pHORF3 were ligated with 35 ng of polished M. hyopneumoniae genomic DNA fragments using 3 U T4 DNA ligase (Promega, Mannheim, Germany) and ligase buffer in 60 μL reaction volume at 16°C overnight. The DNA was precipitated as described above and dissolved in 35 μL dH2O. The DNA solution was mixed with 25 μL of electrocompetent TOP10F′ (Invitrogen, Karlsruhe, Germany), incubated on ice for 1 min and transferred into pre-chilled 0.1 cm electroporation cuvettes. The transformation was performed using a 1.7 kV pulse in a micropulser (Bio-Rad, München, Germany). Immediately, 1 mL pre-

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warmed SOC medium (Sambrook and Russell 2001) was added, and cells were incubated at 37°C and 600 rpm for 1 h. One 10 μL aliquot was used for titration to calculate the amount of independent transformants (Hust et al. 2007). The remaining 990 μL were plated on a 25 cm SOB agar plate with 100 mM glucose and 100 μg/mL ampicillin and incubated overnight at 37°C. The grown colonies were harvested by suspending in 40 mL 2× tryptone yeast (TY) medium with Drigalsky spatula. The library was directly used for phage packaging using Hyperphage, or glycerin was added to a final concentration of 20%, and the library was stored at −80°C in 1 mL aliquots. Enrichment of ORFs using Hyperphage The enrichment of ORFs in the M. hyopneumoniae genomic library required the display of the corresponding polypeptides on phage particles for the panning. Therefore, the library was packaged using Hyperphage which does not encode pIII protein (Rondot et al. 2001; Hust et al. 2006; Soltes et al. 2007). This was done by inoculating 200 mL 2× TY medium containing 100 μg/mL ampicillin and 100 mM glucose with 1 mL of the library stock. The bacteria were grown to an optical density at 600 nm (OD600 of 0.4–0.5 at 37°C and 250 rpm). Twenty five millilitres of the bacterial culture (~1.25×1010 bacteria) were infected with 2.5×1011 Hyperphage, incubated at 37°C for 30 min without shaking, followed by 30 min at 250 rpm. The infected cells were harvested by centrifugation for 10 min at 3,220×g. The pellet was re-suspended in 250 mL 2× TY containing 100 μg/mL ampicillin and 50 μg/mL kanamycin (2× TY-AK). The phages were produced at 30°C and 250 rpm overnight. On the following day, cells were centrifuged for 20 min at 3,220×g. Phage particles in the supernatant were precipitated with 1/5 volume of 20% (w/v) polyethylene glycol (PEG)/ 2.5 M NaCl solution for 1 h on ice with gentle shaking and pelleted by centrifugation for 1 h at 3,220×g at 4°C. The precipitated phages were re-suspended in 300 μL phosphatebuffered saline (PBS; Sambrook and Russell 2001). Residual bacteria and cell debris were removed by additional centrifugation for 5 min at 15,000×g at 20°C. The supernatants containing the polypeptide-expressing phages were stored at 4°C. Phage titration was done according to Hust et al. (2007). Colony PCR E. coli clones bearing pHORF3 were analysed by colony PCR using the primers MHLacZPro_f (5′-GGCTCGTATGT TGTGTGG-3′) and MHgIII_r (5′-GGAAAGACGA CAAAAC TTTAG-3′) and the following protocol: 94°C, 1 min; 56°C, 1 min; 72°C, 2 min; 30 cycles. The DNA was separated by 1% agarose gel electrophoresis.

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SDS-PAGE and immunoblot Phage particles were separated by sodium dodecyl sulfate (SDS) PAGE under reduced conditions according to Laemmli (1970) and blotted onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% (w/v) skimmed milk powder (Roth, Karlsruhe, Germany) in PBST (PBS+0.1% Tween 20) for 1 h at RT. The minor coat protein pIII was detected with mouse anti-pIII monoclonal antibody (mAb; Mobitec, Göttingen, Germany; 1:2,000) for 1.5 h at RT, followed by 2× washing with PBST. Goat antimouse IgG (Fc-specific) antibody conjugated with alkaline phosphatase (Sigma, Taufkirchen, Germany; 1:10,000) was used for detection and visualised by nitro-blue tetrazolium chloride/bromo-4-chloro-3-indolylphosphate toluidine. Selection of phage-encoding immunogenic polypeptides of M.hyopneumoniae Six wells of a MaxiSorb® 96-well microtitre plate (MTP; Nunc, Wiesbaden, Germany) were coated with 150 μL 5 μg/mL goat anti-swine IgG in PBS overnight, washed and blocked with PBST supplemented with 2% (w/v) skim milk powder (2% MPBST) for 1.5 h. In parallel, several wells of a Maxisorb® plate were coated with 150 μL 1×1011 cfu/mL Hyperphage in PBS overnight and blocked with 2% (w/v) M-PBST for 1.5 h. All washing steps between incubation steps were performed three times using PBST buffer and an enzyme-linked immunosorbent assay (ELISA) washer (Tecan Columbus, Crailsheim, Germany) if not otherwise indicated. Porcine convalescent serum (obtained from pigs after infection with M. hyopneumoniae) was diluted 1:10 in PBST, supplemented with 2% MPBST and pre-incubated on Maxisorb® MTP wells coated with Hyperphage for 1 h at RT to remove serum IgG binding to the helperphage. For a second pre-incubation procedure, this step was repeated twice by transferring serum into new Hyperphage-coated wells. After pre-incubation, the swine serum was incubated in goat anti-swine IgG-coated MTP wells for 2 h. After washing, 4×1010 cfu polypeptide phage particles of the Hyperphage-packaged M. hyopneumoniae genomic library were incubated on the captured swine IgGs for 2 h. For the following panning rounds, 1×1012 cfu phage of the previous panning round were used. The non-binding polypeptide phage particles were removed by ten stringent washing steps in the first panning round using an ELISA washer. In the second and third panning round, the number of washing steps was increased to 20 and 30, respectively. Elution of bound phage particles was performed using 200 μL of 10 μg/mL trypsin for 30 min at 37°C. Ten microlitres of the eluted phage solution was used for titration according to Hust et al. (2007). Twenty millilitres of the TOP10F′ E. coli cells was grown to an OD600 of 0.4–0.5, infected with the remaining 190 μL of the eluted phage solution and incubated

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for 30 min at 37°C. Afterwards, the cells were pelleted by centrifugation for 10 min at 3,220×g. The bacterial pellet was dissolved in 250 μL 2× TY medium (1.6% [w/v] tryptone, 1% [w/v] yeast, 0.5% [w/v] NaCl) containing 100 mM glucose and 100 μg/mL ampicillin (2× TY-GA), plated onto 15 cm 2× TYGA agar plates and incubated overnight at 37°C. The grown colonies were harvested in 5 mL 2× TY-GA medium with a Drigalsky spatula. Fifty millilitres of 2× TY-GA medium were inoculated with 200 μL bacteria culture and grown to an OD600 of 0.4–0.5 at 37°C and 250 rpm. Five millilitres of bacterial culture corresponding to about ~2.5×109 cells were infected with 5×1010 cfu Hyperphage, incubated at 37°C for 30 min without shaking and another 30 min with shaking at 250 rpm. The infected cells were harvested by centrifugation for 10 min at 3,220×g. The pellet was re-suspended in 30 mL 2× TY-AK medium, and phages were produced at 30°C and 250 rpm overnight. On the following day, the phage particles were precipitated (see above). Production of individual phage clones for screening Polypropylene 96-well U-bottom plates (Greiner bio-one, Frickenhausen, Germany) containing 175 μL per well 2× TY-GA were inoculated with single E. coli colonies from the phage titration plates of the different panning rounds and incubated at 37°C with constant shaking at 1,000 rpm (thermo shaker PST60-HL4, lab4you, Berlin, Germany) overnight. A new plate with 165 μL 2× TY-GA per well was inoculated with 10 μL of the overnight cultures and incubated at 37°C and 1,000 rpm for 2 h. Afterwards, the bacteria were infected with 5×109 cfu Hyperphage per well and incubated at 37°C without shaking for 30 min, followed by shaking at 1,000 rpm for 30 min. The MTP plate was centrifuged at 3,220×g for 10 min, and the supernatants were discarded. The bacterial pellets were dissolved in 175 μL per well of 2× TY-AK containing 100 mg/mL ampicillin and 30 μg/mL kanamycin and incubated at 30°C at 1,000 rpm overnight for phage production. The bacteria were pelleted as described above, and the supernatants were transferred to a new plate. The phage were precipitated with 1/5 volume 20% PEG/ 2.5 M NaCl solution at 4°C for 1 h and afterwards centrifuged at 3,220×g for 1 h. The phage pellet was dissolved in 150 μL PBS, and residual bacteria were removed by another centrifugation at 3,220×g for 5 min. The phage-containing supernatants were stored at 4°C. Identification of immunogenic polypeptides by monoclonal phage ELISA To capture polypeptide phage particles, 100 μL of 250 ng/mL mouse anti-M13 (B62-FE2, Progen, Freiburg, Germany) in PBS were coated at 4°C overnight. After coating, the wells were blocked with 2% M-PBST. Between each incubation

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step, the wells were washed three times with PBST using an ELISA washer. One hundred fifty μL of the monoclonal phage production were incubated for 2 h. The porcine convalescent serum was diluted 1:100 in 2% MPBST supplemented with 1/10 volume E. coli cell lysate and 1×1010 cfu Hyperphage per millilitre, added to the captured phage particles and incubated for 2 h. The bound porcine IgGs were detected with goat anti-swine IgG conjugated with horseradish peroxidase (HRP; 1:1,000) for 1.5 h and visualised with 3,3′,5,5′-tetramethylbenzidine substrate. The staining reaction was stopped by adding 100 μL 1 N sulphuric acid. The absorbances at 450 nm and scattered light at 620 nm were measured, and the 620 nm value was subtracted using a SUNRISE microtiter plate reader (Tecan, Crailsheim, Germany). Sequence analysis Sequences from selected immunogenic polypeptides were analysed by National Center for Biotechnology Information Blast analysis (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Polypeptide phage production The polypeptide phage were produced in 30 mL scale in shake flask as described by Hust et al. (2007).

Results Construction of pHORF3 The phage vector pHORF3 (Fig. 1) was constructed by replacing the NotI restriction sites in pHORF2 (Hust et al. 2006) with a unique PmeI site in order to allow the introduction of blunt end genomic DNA fragments between

Fig. 2 Analysis of randomly selected clones before (a) and after (b) ORF enrichment by PCR. C Control PCR with pHORF3 (268 bp PCR product), M marker

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the 5′ pelB signal peptide and 3′ gIII gene fragment. If signal peptide encoding DNA, the inserted genomic DNA fragment and gIII form an intact ORF, the encoded peptide is displayed as gIII fusion protein on the phage surface (Fig. 1). The second domain of the previously identified immunogenic protein Mhp651 of M. hyopneumoniae strain 232 encoded on the vector pMhp651-501 (Meens et al. 2006) was cloned into the PmeI site of pHORF3 and used as a positive control for the ELISA detection. Construction of the M. hyopneumoniae genome library and enrichment of ORFs Genomic DNA of M. hyopneumoniae was sheared by sonication, polished with T4 DNA polymerase and Klenow fragment and afterwards cloned into the PmeI site of pHORF3. The total scheme of cloning and panning is shown in Fig. 1. The library was determined to contain 1.4×105 independent clones. After cloning into pHORF3, about 90% of the clones of the M. hyopneumoniae genomic library contained DNA inserts of 50–700 bp as analysed by colony PCR (Fig. 2a). After enrichment of ORFs in the M. hyopneumoniae genomic library by packaging with Hyperphage (Rondot et al. 2001; Soltes et al. 2007), the size of DNA inserts decreased to between 50 and 300 bp as determined by re-infection into E. coli and colony PCR (Fig. 2b). DNA sequencing of 46 clones revealed that 69% of the clones contained an ORF consisting of a M. hyopneumoniae genomic DNA fragment without stop codons and positioned in frame with the pelB leader and gIII ORF. The minimum insert size determined by sequencing was 46 bp, and the maximum size was 318 bp with an average insert size of 142 bp. Therefore, the library coverage after packaging is about 20 fold. In addition, the Hyperphagepackaged genomic library and six randomly selected and re-packaged clones were analysed by SDS-PAGE under reducing conditions, and blotted onto PVDF membranes. Then,

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phage increased three to four orders of magnitude compared to the first panning round. Identification of immunogenic polypeptides

Fig. 3 Immunoblot of randomly picked polypeptide phage clones from the Hyperphage packaged ORF-enriched library (lanes 1–6), the ORF-enriched library (lane L) and Hyperphage (lane H). Twentymicrolitre samples of polypeptide phage clones (~1×1010), 1×109 phage particles of the ORF-enriched library and 1×1010 Hyperphage were boiled under reducing conditions and separated on a 10% SDSPAGE. After Western blot, the PVDF membrane was stained using mouse mAb anti-pIII. Wildtype pIII and pIII::oligopeptide fusionprotein bands are indicated by arrows

pIII was visualised by immunostaining using a monoclonal mouse anti-pIII antibody (Fig. 3). Protein pIII has a calculated molecular mass of 42.5 kDa, but it runs at an apparent molecular mass of 65 kDa in SDS-PAGE (Goldsmith and Konigsberg 1977; Breitling et al. 1991) as shown by the Hyperphage negative control in Fig. 3. All six analysed single clones of the ORF-enriched library show a wild type pIII band and a clear pIII fusion protein band, whereas only polypeptide::pIII fusion protein was detectable in the Hyperphagepackaged ORF-enriched M. hyopneumoniae genomic library. Selection of immunogenic polypeptides The panning procedure for identifying immunogenic polypeptides of M. hyopneumoniae was performed in MTPs using convalescent serum derived from pigs after an M. hyopneumoniae infection (see scheme in Fig. 1). Preliminary studies had revealed that porcine serum mediates unspecific phage binding inhibiting the specific isolation of immunogenic polypeptides encoded by phage libraries (data not shown). Therefore, it was critical to perform a pre-incubation of the porcine convalescent serum in Hyperphage-coated microtitre wells to remove phage-binding IgGs from the serum to be used for the panning procedure. Two slightly different pre-incubation procedures were performed. In the first procedure, the convalescent serum was pre-incubated once and in the second approach three times. After preincubation of the convalescent serum, IgG was captured in MTPs coated with goat anti-swine IgG. Subsequently, the Hyperphage-packaged ORF-enriched genomic M. hyopneumoniae library was incubated on the captured porcine serum IgG, and non-binding phage particles were removed by stringent washing. Binders were eluted, amplified in E. coli and packaged again with Hyperphage before starting another panning round. In total, up to three panning rounds were performed. The titre after each panning round was determined (Fig. 4a). In the third panning round, the titre of eluted

E. coli clones infected by the eluted phage were isolated from panning rounds 1, 2 and 3 of two independent panning experiments and used for monoclonal phage production in MTPs and screening by phage ELISA to identify phage displaying immunogenic polypeptides. Since direct coating of the polypeptide phage particles led to increased background (data not shown), MTPs were coated with mouse anti-pVIII antibody for indirect coating by capturing phage particles from the suspension. Afterwards, convalescent serum was added, and captured phage expressing immunogenic M. hyopneumoniae peptides were detected using anti-swine IgG conjugate. In contrast to bovine and human serum, porcine serum generally caused high background in ELISA and immunoblot assays (data not shown). To reduce this unspecific background signal, the porcine convalescent serum was diluted in PBS supplemented with skim milk, E. coli cell lysate and Hyperphage particles. An example for the monoclonal phage ELISA is given in Fig. 4b. The Mhp651 phage containing an immunogenic polypeptide of M. hyopneumoniae was used as the positive control and an anti-CRP scFv phage particle as negative control. Interestingly, Hyperphage alone caused a lower background signal than phage particles displaying a nonrelevant polypeptide demonstrating the importance to use appropriate phage as negative control (data not shown). Thirty clones out of 540 analysed clones, derived from 90 clones per panning round of both pannings, were positive in ELISA and were subjected to DNA sequencing. Six individual polypeptides were identified by NCBI Blast analysis (Table 1). A correlation between the strength of the measured ELISA signal and the frequency of identical clones isolated after panning was found. Phage clone JOK55-IE3 displaying a polypeptide fragment of P97 gave the highest ELISA signals corresponding to 13 isolated clones in comparison to phage displaying transcription elongation factor (JOK60-IIIA10) or H5 (JOK60-IID7) with six or four identical isolated clones, respectively. The ELISA signals obtained from phage clones displaying the other three identified polypeptides were only lean over the threshold which is corresponding with the low frequency of two to three isolated identical clones. Verification of the immunogenic character of the selected polypeptides The immunogenic character of the six selected polypeptides was verified by phage titration ELISA using re-convalescent serum and negative serum from a non-infected pig (Fig. 5). Five of six selected immunogenic polypeptides were detected by the re-convalescent serum. The ELISA signals of

454 Fig. 4 The selection of immunogenic polypeptides. a Comparison of the antibody phage titres (cfu) obtained from each panning round after elution. b ELISA with monoclonal oligopeptide phage clones from the third panning round produced in MTPs. For phage capturing, the wells were coated with 250 ng/mL mouse antipVIII IgG. Monoclonal phage particles of 150 μL (~1× 108 cfu) were bound, which were detected by convalescence sera (1:100) obtained from a pig previously infected with M. hyopneumoniae. Bound swine antibodies were detected using goat anti-swine HRP conjugate (1:1,000). As negative control, anti-CRP scFv phage particles (1.5×1010 cfu per well) and, as positive control, Mhp651 oligopeptide phage particles (1.5× 1010 cfu/well) were used. The threshold is marked with a horizontal line. Only clones with a signal above the threshold were subjected to further analysis

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A

B

. . . .

. . . .

JOK60-IIA12 were comparable to the ELISA signals of the Hyperphage control; therefore, the immunogenic character of this polypeptide was not verified. The negative serum did not bind to any of the immunogenic polypeptides. The Hyperphage control was slightly more strongly bound by the reconvalescent serum as by the negative serum. JOK55-IE3 displaying P97 was best detected by the re-convalescent serum. When high-phage titres were captured, i.e. greater than 1×109 cfu per well or 1×1010 cfu per millilitre, the signal-tonoise ratio was improved compared to the ELISA using MTP produced polypeptide phage particles.

Discussion The development and enhancement of genome libraries obtained by phage display could be a crucial tool to identify those immunogenic polypeptides which cannot be identified

by proteome-based methods. So far, there have been described only rare studies using genomic phage display libraries (Beghetto et al. 2006; Nilsson et al. 2004; Hertveldt et al. 2003); mainly, cDNA-based libraries were used for this purpose (Cochrane et al. 2000; Govarts et al. 2007; Rhyner et al. 2004; Yang et al. 2007). For the application of genomic phage display libraries, the enrichment of ORFs and the presentation of the corresponding polypeptides on M13 phage are essential. After cloning genomic DNA fragments into phage display vectors, less than 6% of the phagemids contains a complete ORF consisting of a pelB leader, cloned DNA insert and gIII according to theoretical calculations. As a result, a subsequent panning would be performed with a phage library containing more than 90% of “junk clones.” Since each phage display selection based on the ability to distinguish single molecular binding events in a background of 108–1011 “unspecific” molecules, a library with greater than 90% “junk” sequences dramatically ham-

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Table 1 Overview of the immunogenic polypeptides identified in M. hyopneumoniae M. hyopneumoniae strain J Clone

Insert (bp) Clone frequency Base pair genome segment

Identity to other strains

Protein ID (NCBI) Gene product

JOK55-IE3

187

13

216986–217172 AAZ44285.1

JOK60-IIIA10

118

6

883865–883982 AAZ44750.1

JOK55-IB7

166

2

532752–532587 AAZ44523.2

JOK55-IG1

172

3

200862–200691 AAZ44260.1

JOK60-IID7

121

4

871122–871002 AAZ44745.2

JOK60-IA12

43

2

646634–646592 AAZ44588.1

Putative protein P97 Transcription elongation factor GreA Conserved hypothetical protein Methionine aminopeptidase Hypothetical protein H5 Putative lipoprotein

Locus tag

Strain 232 Strain 7448 locus tag locus (% identity) tag (% identity)

MHJ_0194 mhp183 (85%) MHJ_0667 mhp690 (100%)

MHP7448_0198 (85%) MHP7448_0668 (100%)

MHJ_0437 mhp443 (96%)

MHP7448_0439 (100%)

MHJ_0169 mhp209 (100%) MHJ_0662 mhp683 (97%) MHJ_0501 mhp502 (100%)

MHP7448_0173 (98%) MHP7448_0662 (96%) MHP7448_0504 (100%)

The protein IDs and locus tags are given for M. hyopneumoniae strain J (NCBI GenBank AE017243). In addition, locus tags and percent identity to strain J are shown for M. hyopneumoniae strain 232 (NCBI GenBank AE017332) and strain 7448 (AE017244).

pers the success of the panning. Therefore, a M13 phage display system is required which simultaneously facilitates the enrichment of ORFs and the display of the corresponding polypeptide on the phage surface without additional subcloning steps. In contrast to ORF libraries generated from E. coli genome fragments, which were tested in parallel resulting in more than 1×106 independent clones upon a single transformation

(data not shown), the cloning of the M. hyopneumoniae genomic library was hampered by about 1,000-fold lower transformation rates. Furthermore, it was hypothesized that the low GC content of about 29% may cause an incomplete polishing of the fragmented DNA by T4 DNA polymerase (Sirand-Pugnet et al. 2007a, b). Therefore, an additional polishing step of the genomic DNA fragments was performed using a Klenow fragment, which dramatically improved the

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.

Fig. 5 Verification of the immunogenic character of the selected polypeptides. ELISA with a serial dilution of the selected polypeptide phage clones produced in a 30 mL scale. For phage capturing, the wells were coated with 250 ng/mL mouse anti-pVIII IgG. Monoclonal phage particles (100 μL) were bound and detected by convalescence sera

.

.

.

(1:200) obtained from a pig previously infected with M. hyopneumoniae or negative serum (1:200) from a non-infected pig. Bound swine antibodies were detected using goat anti-swine HRP conjugate (1:2,000). As positive control Mhp651 oligopeptide phage particles were used

456

generation of Mycoplasma libraries resulting in a maximum of 1.4×105 independent clones upon a single transformation. This two-step polishing procedure was also described by Margulies et al. (2005) for the cloning of M. genitalium genomic DNA fragments. The average size of the cloned insert decreased after Hyperphage packaging. This decrease in the insert size could be attributed to three effects. In general, phagemids containing a short insert will be propagated faster in E. coli as phagemids with longer insert. Usually, pIII fused with shorter polypeptides will be better produced leading to an increased phage particle production. Furthermore, longer mycoplasma gene fragments statistically contain more opal stop codons hampering the expression of the pIII fusion protein and Hyperphage-based phage production in E. coli. After packaging, the rate of ORF enrichment was analyzed by DNA sequencing showing that 69% of the clones contained a continuous ORF. The remaining 31% of the clones did not contain an insert, the inserts were out of frame with gIII or they were in frame but have a stop codon in the genomic mycoplasm DNA insert. This ORF enrichment of a genome library is in accordance with an earlier study using the phagemid pHORF2 and Hyperphage for the selection of oligopeptides (Hust et al. 2006) Although Zacchi et al. (2003) or Faix et al. (2004) described a higher enrichment of ORFs by cloning the DNA of interest in fusion with a resistance marker, the resistance marker has to be removed from the ORF-enriched library by subsequent cloning and transformation steps causing a decrease in library diversity. Moreover, the proportion of intact ORFs in the library also reduced to 70% (Faix et al. 2004), which is comparable to our study. However, in contrast to the resistance markerbased systems, the Hyperphage-based ORF enrichment does not require additional cloning steps and, therefore, facilitates the generation of highly diverse libraries which can be directly used for the antigen selection procedure using convalescent sera. Preliminary experiments revealed that the pre-incubation of the convalescent pig sera on Hyperphage before panning and the addition of Hyperphage particles and E. coli lysate into the blocking buffer during the panning procedure were absolutely required to reduce background binding and to allow a specific selection of phage particles according to the displayed polypeptide encoded by M. hyopneumoniae ORFs. Unspecific binding of M13 phage has been demonstrated for many sera (Eshaghi et al. 2005) and is probably caused by previous contact and immunisation with these E. coli phages. The signal-to-noise ratio was optimised by using purified phage particles instead of MTP-produced phage particles directly used from the supernatant. In this study, six immunogenic polypeptides were selected from the genomic phage display library by panning on captured convalescent serum. The immunogenic character of the selected polypeptides was verified by phage titration ELISA

Appl Microbiol Biotechnol (2008) 80:447–458

using re-convalescent serum from infected pigs and negative serum from a non-infected pig. The most frequently isolated and re-convalescent serum best-bound phage displaying P97 (MHJ_0194, clone JOK55-IE3) is essential for the adherence of M. hyopneumoniae to ciliated epithelial cells of the porcine respiratory tract (Hsu and Minion 1998a). P97 is a membrane protein containing two repetitive elements within the C-terminal portion, designated R1 and R2. R1 has been identified as the cilium-binding epitope. P97 is an immunodominat protein, and especially, the R1 repeat structure is recognized by convalescent swine serum (Minion et al. 2000). The DNA insert in clone JOK55-IE3 covers the complete R1 region (amino acid position 813–874), including nine copies of the repetitive sequence (AAKPV/E). This domain has already been used in experimental recombinant vaccines, inducing the production of systemic and mucosal IgA antibodies in mice (Conceição et al. 2006) and pigs (Minion et al. 2004). Protein MHJ_0662 (clone JOK60-IID7) is postulated as a hypothetical virulence factor having a variable number of amino acid tandem repeats in different M. hyopneumoniae strains (Ferreira and De Castro 2007). It is a membrane protein and belongs to the family of P102 paralogs (Adams et al. 2005). The genes encoding P102 (MHJ_0195) and P97 (MHJ_0194) are organized as a two-gene operon (Hsu and Minion 1998b). The protein P102 is supposed to function in adherence either directly or indirectly by supporting P97 activity. In all three M. hyopneumoniae strains sequenced up to now, the genome revealed up to six paralogs of both the P97 and P102 genes (Minion et al. 2004; Vasconcelos et al. 2005). The in vivo expression of these paralogous protein families has been shown in M. hyopneumoniae 232 (Adams et al. 2005). MJH_0437 (JOK55-IB7) is a conserved hypothetical membrane protein which shows moderate homology to different hypothetical membrane from other mycoplasma species, like Mycoplasma capricolum subsp. capricolum ATCC 27343(MCAP_0347; acc. no. YP_424331), Mycoplasma synoviae strain 53 (MS53_0462; acc. no. YP_278582) and Mycoplasma mycoides subsp. mycoides SC PG1T (MSC_0633, acc. no. NP_975613). None of these proteins, including MHJ0437, has been functionally characterized, so far. The methionine aminopeptidase MHJ_0169 (clone JOK55-IG1) is an essential cytoplasmatic enzyme involved in protein N-terminal methionine cleavage to generate mature proteins. Immunogenic properties or any virulence-associated function have not been described neither for the methionine aminopeptidase nor for the transcription elongation factor GreA (MHJ0667, clone JOK60-IIIA10). It was not possible to verify the immunogenic character of the putative lipoprotein MHJ_0501 (clone JOK60-IA12) by titration ELISA. The panning of the Hyperphage based ORF-enriched library of M. hyopneumoniae genomic DNA fragments to convalescent pig sera resulted in the identification of two already known immunogenic polypeptides and of three

Appl Microbiol Biotechnol (2008) 80:447–458

immunogenic polypeptides, which have not been identified, so far. In contrast to genomic or cDNA libraries containing full-length genes, the enrichment of small but continuous ORFs in the library after packaging with Hyperphage is highly advantageous for mycoplasmal systems, because they encode one stop codon as tryptophan whereas E. coli does not. For example, immunogenic proteins like P97 could not be identified by full cDNA cloning due to the occurrence of several of these tryptophan-encoding stop codons. The restriction to smaller polypeptides and E. coli’s Sec pathway by the used phage display system might not be able to represent the whole spectrum of immungenic epitopes of M. hyopneumoniae antigens. The loss of complex antigens with structural epitopes as well as of antigens which cannot be secreted into the periplasm of E. coli via the Sec pathway, like membrane or cytosolic proteins, have to be considered. However, in this study, the Hyperphage-based ORF-enrichment and phage display system already resulted in the identification of one lipoprotein (MHJ_0502), three membrane proteins (MHJ_0194, MHJ_0437 and MHJ_0662) and two intracellular proteins (MHJ_0667 and MHJ_0169) representing different types of proteins. Presumably, the display of the smaller polypeptide is much more independent of the folding and secretion apparatus in E. coli and should be less problematic regarding the Sec pathway required for this phage display system. But, the detection and selection by convalescent sera can by limited to serum antibodies recognizing linear epitopes. A combination of phagemid vectors offering the secretion via the twin-arginin translocation (Paschke and Höhne 2005) and signal recognition particle (Valent 2001) pathway in addition to the Sec pathway could enhance the display of a broader spectrum of polypeptide antigens. In the future, the identified immunogenic polypeptides will be evaluated for their use in veterinary diagnostics for detection of M. hyopneumoniae infections in pigs and their potential as vaccines.

Acknowledgements We would like to thank Stefan Dübel for corrections and discussion on the manuscript and Saskia Helmsing for technical assistance.

References Adams C, Pitzer J, Minion FC (2005) In vivo expression analysis of the P97 and P102 paralog families of Mycoplasma hyopneumoniae. Infect Immun 73:7784–7787 Beghetto E, Gargano N, Ricci S, Garufi G, Peppoloni S, Montagnani F, Oggioni M, Pozzi G, Felici F (2006) Discovery of novel Streptococcus pneumoniae antigens by screening a whole-genome lambda-display library. FEMS Microbiol Lett 262:14–21 Breitling F, Dübel S, Seehaus T, Klewinghaus I, Little M (1991) A surface expression vector for antibody screening. Gene 104:147–153

457 Cochrane D, Webster C, Masih G, McCafferty J (2000) Identification of natural ligands for SH2 domains from a phage display cDNA library. J Mol Biol 297:89–97 Conceição FR, Moreira AN, Dellagostin OA (2006) A recombinant chimera composed of R1 repeat region of Mycoplasma hyopneumoniae P97 adhesin with Escherichia coli heat-labile enterotoxin B subunit elicits immune response in mice. Vaccine 24:5734–5743 Crameri R, Kodzius R, Konthur Z, Lehrach H, Blaser K, Walter G (2001) Tapping allergen repertoires by advanced cloning technologies. Int Arch Allergy Immunol 124:43–47 Delvecchio VG et al (2006) Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Appl Environ Microbiol 72:6355–6363 Eshaghi M, Tan WS, Yusoff K (2005) Identification of epitopes in the nucleocapsid protein of Nipah virus using a linear phage-displayed random peptide library. J Med Virol 75:147–152 Faix PH, Burg MA, Gonzales M, Ravey EP, Baird A, Larocca D (2004) Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection. Biotechniques 36:1018–1022 Ferreira HB, De Castro LA (2007) A preliminary survey of M. hyopneumoniae virulence factors based on comparitive genomic analysis. Gen Mol Biol 30:245–255 Futo S, Seto Y, Mitsuse S, Mori Y, Suzuki T, Kawai K (1995) Molecular cloning of a 46-kilodalton surface antigen (P46) gene from Mycoplasma hyopneumoniae: direct evidence of CGG codon usage for arginine. J Bacteriol 177:1915–1917 Gibson DG et al (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319:1215–1220 Glass JI, Assad-Garcia N, Alperovich N, Yooseph S, Lewis MR, Maruf M, Hutchison CA, Smith HO, Venter JC (2006) Essential genes of a minimal bacterium. Proc Natl Acad Sci USA 103:425–430 Goldsmith ME, Konigsberg WH (1977) Adsorption protein of the bacteriophage fd: isolation, molecular properties, and location in the virus. Biochemistry 16:2686–2694 Govarts C, Somers K, Hupperts R, Stinissen P, Somers V (2007) Exploring cDNA phage display for autoantibody profiling in the serum of multiple sclerosis patients: optimization of the selection procedure. Ann N Y Acad Sci 1109:372–384 Hertveldt K, Dechassa ML, Robben J, Volckaert G (2003) Identification of Gal80p-interacting proteins by Saccharomyces cerevisiae whole genome phage display. Gene 307:141–149 Hoelzle LE, Hoelzle K, Harder A, Ritzmann M, Aupperle H, Schoon H, Heinritzi K, Wittenbrink MM (2007) First identification and functional characterization of an immunogenic protein in unculturable haemotrophic Mycoplasmas (Mycoplasma suis HspA1). FEMS Immunol Med Microbiol 49:215–223 Hsu T, Minion FC (1998a) Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin. Infect Immun 66:4762–4766 Hsu T, Minion FC (1998b) Molecular analysis of the P97 cilium adhesin operon of Mycoplasma hyopneumoniae. Gene 214:13–23 Hsu T, Artiushin S, Minion FC (1997) Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae. J Bacteriol 179:1317–1323 Huntley JF, Conley PG, Hagman KE, Norgard MV (2007) Characterization of Francisella tularensis outer membrane proteins. J Bacteriol 189:561–574 Hust M, Dübel S (2004) Mating antibody phage display with proteomics. Trends Biotechnol 22:8–14 Hust M, Meysing M, Schirrmann T, Selke M, Meens J, Gerlach G, Dübel S (2006) Enrichment of open reading frames presented on bacteriophage M13 using hyperphage. Biotechniques 41:335–342 Hust M, Dübel S, Schirrmann T (2007) Selection of recombinant antibodies from antibody gene libraries. Methods Mol Biol 408:243– 255

458 Jacobsen ID, Meens J, Baltes N, Gerlach G (2005) Differential expression of non-cytoplasmic Actinobacillus pleuropneumoniae proteins induced by addition of bronchoalveolar lavage fluid. Vet Microbiol 109:245–256 Kim MF, Heidari MB, Stull SJ, McIntosh MA, Wise KS (1990) Identification and mapping of an immunogenic region of Mycoplasma hyopneumoniae p65 surface lipoprotein expressed in Escherichia coli from a cloned genomic fragment. Infect Immun 58:2637–2643 Kodzius R, Rhyner C, Konthur Z, Buczek D, Lehrach H, Walter G, Crameri R (2003) Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays. Comb Chem High Throughput Screen 6:147–154 Konthur Z, Hust M, Dübel S (2005) Perspectives for systematic in vitro antibody generation. Gene 364:19–29 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 Lartigue C, Glass JI, Alperovich N, Pieper R, Parmar PP, Hutchison CA, Smith HO, Venter JC (2007) Genome transplantation in bacteria: changing one species to another. Science 317:632–638 Maas A, Meens J, Baltes N, Hennig-Pauka I, Gerlach G (2006) Development of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae infection. Vaccine 24:7226–7237 Madsen ML, Puttamreddy S, Thacker EL, Carruthers MD, Minion FC (2008) Transcriptome changes in Mycoplasma hyopneumoniae during infection. Infect Immun 76:658–663 Margulies M et al (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376–380 McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554 Meens J, Selke M, Gerlach G (2006) Identification and immunological characterization of conserved Mycoplasma hyopneumoniae lipoproteins Mhp378 and Mhp651. Vet Microbiol 116:85–95 Minion FC, Adams C, Hsu T (2000) R1 region of P97 mediates adherence of Mycoplasma hyopneumoniae to swine cilia. Infect Immun 68:3056–3060 Minion FC, Lefkowitz EJ, Madsen ML, Cleary BJ, Swartzell SM, Mahairas GG (2004) The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J Bacteriol 186:7123–7133 Nilsson M, Bjerketorp J, Wiebensjö A, Ljungh A, Frykberg L, Guss B (2004) A von Willebrand factor-binding protein from Staphylococcus lugdunensis. FEMS Microbiol Lett 234:155–161

Appl Microbiol Biotechnol (2008) 80:447–458 Paschke M, Höhne W (2005) A twin-arginine translocation (Tat)mediated phage display system. Gene 350:79–88 Rhyner C, Weichel M, Flückiger S, Hemmann S, Kleber-Janke T, Crameri R (2004) Cloning allergens via phage display. Methods 32:212–218 Rondot S, Koch J, Breitling F, Dübel S (2001) A helper phage to improve single-chain antibody presentation in phage display. Nat Biotechnol 19:75–78 Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New York Sellman BR, Howell AP, Kelly-Boyd C, Baker SM (2005) Identification of immunogenic and serum binding proteins of Staphylococcus epidermidis. Infect Immun 73:6591–600 Sidhu SS, Koide S (2007) Phage display for engineering and analyzing protein interaction interfaces. Curr Opin Struct Biol 17:481–487 Sirand-Pugnet P, Citti C, Barré A, Blanchard A (2007a) Evolution of mollicutes: down a bumpy road with twists and turns. Res Microbiol 158:754–766 Sirand-Pugnet P et al (2007b) Being pathogenic, plastic, and sexual while living with a nearly minimal bacterial genome. PLoS Genet 3:e75 Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317 Soltes G, Hust M, Ng KKY, Bansal A, Field J, Stewart DIH, Dübel S, Cha S, Wiersma EJ (2007) On the influence of vector design on antibody phage display. J Biotechnol 127:626–637 Strasser M, Frey J, Bestetti G, Kobisch M, Nicolet J (1991) Cloning and expression of a species-specific early immunogenic 36kilodalton protein of Mycoplasma hyopneumoniae in Escherichia coli. Infect Immun 59:1217–1222 Taussig MJ et al (2007) ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome. Nat Methods 4:13–17 Valent QA (2001) Signal recognition particle mediated protein targeting in Escherichia coli. Antonie Van Leeuwenhoek 79:17–31 Vasconcelos ATR et al (2005) Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae. J Bacteriol 187:5568–5577 Yang E, Shim JS, Woo H, Kim K, Kwon HJ (2007) Aminopeptidase N/CD13 induces angiogenesis through interaction with a proangiogenic protein, galectin-3. Biochem Biophys Res Commun 363:336–341 Zacchi P, Sblattero D, Florian F, Marzari R, Bradbury ARM (2003) Selecting open reading frames from DNA. Genome Res 13:980–990