Efficient method for construction comprehensive murine Fab antibody libraries displayed on phage (original) (raw)
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The New Microbiologica: official journal of the Italian Society for Medical Virology (SIVIM)
A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III and light chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide, whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was used to generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragment antibody directed against the nucleoprotein (NP) ...
Research in Virology, 1997
Phagemid pComb3 is a widely used vector for molecular cloning of the antibody repertoire and for production of phage display libraries. However, in practical use, the utilization of this vector has some drawbacks. In this work we describe the construction of pComb3/TIG, an improved, easily manipulated vector for the cloning and display of antibody fragment libraries on the surface of filamentous phage. The two small "stuffer" fragments at the cloning sites were replaced with long DNA fragments, for easier differentiation of the correctly cut forms of the vector. Moreover, in pComb3/TIG the fragment at the heavy-chain-fragment cloning site contains an acid phosphatase-encoding gene. This feature allows the easy distinction of the Escherichia colicells containing the unmodified form of the phagemid instead of the heavy-chain fragment coding cDNA in a simple plate hi&chemical assay.
Rabbit monoclonal Fab derived from a phage display library
Journal of Immunological Methods, 1998
Ž. Rabbit monoclonal antibodies RmAb are not routinely obtained by eukaryotic cell fusion techniques. Therefore, we have applied phage display technology to produce a recombinant rabbit Fab molecule directed against the KLH model antigen. The Fab fragments selected from the rabbit phage display library were subcloned in an expression vector to permit Ž. the production of a fusion protein comprising a dimer of bacterial alkaline phosphatase phoA. This fusion protein was directly produced into the periplasmic space of Escherichia coli. We show that a crude extract containing these conjugates can be used in a direct enzyme immunoassay, as exemplified in the case of the KLH antigen.
A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-delta geneIII expression, primer usage for PCR amplification of variable region genes, scFv assembly strategy and subsequent directional cloning using a single rare cutting restriction enzyme. This integrated cloning, screening and selection system allowed us to rapidly obtain antigen binding scFvs derived from spleen-cell repertoires of mice immunized with ampicillin as well as from all hybridoma cell lines tested to date. As representative examples, cloning of monoclonal antibodies against a his tag, leucine zippers, the tumor marker EGP-2 and the insecticide DDT is presented. Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. These contaminating variable region genes were successfully eliminated by employing the optimized phage display system, thus avoiding time consuming sequencing of non-binding scFv genes. To maximize soluble expression of functional scFvs subsequent to cloning, a compatible vector series to simplify modification, detection, multimerization and rapid purification of recombinant antibody fragments was constructed.
Gene, 1993
We have combined the high cloning efficiency of the h bacteriophage vectors with the surface expression screening method for the display of combinatorial antibody fragment (Fab) libraries on the surface of filamentous phage particles. The utility of the herein described ImmunoZAP TM 13 system for the isolation of Fabs that specifically bind antigen is demonstrated using two phagemid display libraries prepared from a previously characterized human combinatorial library. The percentage of clones that specifically bind antigen is maintained throughout the process of subcloning the LC and I$, genes into ImmunoZAP 13, in vivo mass excision to convert the h library to a phagemid library, and preparation of phagemid particles displaying Fabs. Specific phagemid were isolated from libraries containing 0.6% and 0.03% tetanus toxoid (TT)-binding clones after two and three rounds of biopanning, respectively. Relative binding curves determined on a small sample of isolated clones indicate that several unique immunoglobulin Fab fragments have been isolated.
Display of Expression Products of cDNA Libraries on Phage Surfaces
European Journal of Biochemistry, 2008
Techniques for cloning cDNAs from bacteriophage libraries immobilised on solid supports are well established. However, these techniques do not allow selective enrichment of clones expressing proteins of interest. Screening of cDNA libraries would be simplified if the proteins encoded by cDNAs could be expressed on the surface of phage. Phage carrying genes encoding proteins for which a ligand is available can be selected directly by affinity interaction [Crameri, R. & Suter, M. (1993) Gene (Amst.) 137, 69-75]. The expression products from a cDNA library from Aspergillus fumigatus have been displayed on the surface of the filamentous phage M13 and screened for gene products binding to human serum IgE. The physical linkage of cDNA-encoded proteins to the genetic information required for their production, achieved by exploiting the high-affinity interaction of the Jun and Fos leucine zippers, allows screening of up to 1 X 10" independent clones in 50-yl aliquots applied to a well of a microtiter plate coated with the ligand. Phage displaying IgE-binding proteins were selectively enriched lo5-106-fold over non-specific phage after six rounds of growth and selection. The apparent molecular mass of the proteins selected from the cDNA library was in the range 20-40 kDa. Restriction enzyme analysis and preliminary sequence determination of 12 selected inserts revealed different sequences. The ability of the proteins to bind to human serum IgE was corroborated by enzyme-linked immunosorbent assay and by Western-blot analysis. The developed cloning strategy allows isolation of cDNAs encoding proteins for which a ligand is available and circumvents immobilisation of the libraries on solid-phase supports which hamper selective enrichment of clones expressing the desired protein.
Phage-display libraries of murine and human antibody Fab fragments
Molecular Biotechnology, 1996
We provide efficient and detailed procedures for construction, expression, and screening of compi~6hensire libraries of murine or human antibody Fab fragments displayed on the surface Of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab phages from libraries, and for selecting antigen binders by Panning are presented. Thetatter protocol includesa~procedtire for trypsin elution of bound phage.
Assembly of combinatorial antibody libraries on phage surfaces: the gene III site
Proceedings of the National Academy of Sciences, 1991
A phagemid system was developed for the monovalent display of combinatorial antibody Fab libraries on the surface of filamentous phage M13. Fab fragments were fused to the carboxyl-terminal domain of the gene III protein. Phage displaying Fab fragments on their surface, or Phabs, were enriched by 10(3)- to 10(5)-fold on antigen-coated surfaces over nonspecific phage. The method may replace current antibody cloning techniques.