Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein

The major vault protein is the main component on multimeric vault particles, that are likely to play an essential role in normal cell physiology and to be associated with multidrug resistance of tumour cells. In order to unravel the function of vaults and their putative contribution to multidrug resistance, specific antibodies are invaluable tools. Until now, only conventional major vault protein-reactive murine monoclonal antibodies have been generated, that are most suitable for immunohistochemical analyses. The phage display method allows for selection of human antibody fragments with potential use in clinical applications. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human Fab fragments recognising major vault protein we used a large non-immunized human Fab fragment phage library. Phages displaying major vault protein-reactive Fabs were obtained through several rounds of selection on major vault protein-coated immunotubes and subsequent amplification in TG1 E coli bacteria. Eventually, one major vault protein-reactive clone was selected and further examined. The anti-major vault protein Fab was found suitable for immunohistochemical and Western blot analysis of tumour cell lines and human tissues. BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs. The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies. Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms

PuLSE:Quality control and quantification of peptide sequences explored by phage display libraries

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use

Analyzing ligation mixtures using a PCR based method

We have developed a simple and effective method (Lig-PCR) for monitoring ligation reactions using PCR and primers that are common to many cloning vectors. Ligation mixtures can directly be used as templates and the results can be analyzed by conventional gel electrophoresis. The PCR products are representative of the recombinant molecules created during ligation and the corresponding transformants. Orientation of inserts can also be determined using an internal primer. The usefulness of this method has been demonstrated using ligation mixtures of two cDNA’s derived from the salivary glands of Aedes aegypti mosquitoes. The method described here is sensitive and easy to perform compared to currently available methods

Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas

SC142-reactive antigen are highly glycosylated glycoproteins expressed on tissues of gastric and colon cancers but not on normal tissues. Murine SC142 antibody specific for the SC142-reactive antigen has been produced by immunisation with SNU16 stomach cancer cells. However, SC142 antibody has several potential problems such as high immunogenicity and poor tumour penetration owing to their large size. To improve tumour penetration potential in vivo, recombinant single-chain fragments have been produced using the original hybridoma cells as a source of variable heavy- and variable light-chain-encoding antibody genes. The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry. Analysis by DNA sequencing, SDS–PAGE and Western blotting has demonstrated the integrity of the single-chain fragments. Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody. BIAcore biosensor binding experiments showed that the SC142 single-chain fragments had an ideal dissociation rate constant as a tumour imaging reagent. These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent

Antibody Engineering Using Phage Display with a Coiled-Coil Heterodimeric Fv Antibody Fragment

A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to VH and VL for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of VH frameworks and VH-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering

Expanding the Versatility of Phage Display I: Efficient Display of Peptide-Tags on Protein VII of the Filamentous Phage

Background: Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display. Methodology/Principal Findings: Here we demonstrate how standard pIII or pVIII display phagemids are complemented with a helper phage which supports production of virions that are tagged with octa FLAG, HIS6 or AviTag on pVII. The periplasmic signal sequence required for pIII and pVIII display, and which has been added to pVII in earlier studies, is omitted altogether. Conclusions/Significance: Tagging on pVII is an important and very useful add-on feature to standard pIII and pVII display. Any phagemid bearing a protein of interest on either pIII or pVIII can be tagged with any of the tags depending simply on choice of helper phage. We show in this paper how such tags may be utilized for immobilization and separation as well as purification and detection of monoclonal and polyclonal phage populations. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Expanding the Versatility of Phage Display II: Improved Affinity Selection of Folded Domains on Protein VII and IX of the Filamentous Phage

Background: Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display. Methodology/Principal Findings: Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient. Conclusions/Significance: Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Selection of Anti-Sulfadimidine Specific ScFvs from a Hybridoma Cell by Eukaryotic Ribosome Display

BACKGROUND:Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes. METHODOLOGY/PRINCIPAL FINDINGS:In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM(2)) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA-ribosome-antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM(2)-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM(2) by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis. CONCLUSIONS/SIGNIFICANCE:The selection of anti-SM(2) specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs