Cloning in a bacteriophage lambda vector for the display of binding proteins on filamentous phage.

We have combined the efficiency and ease of use of bacteriophage lambda vectors with the power of phage display screening technology to create SurfZAP. The use of bacteriophage lambda allows the construction of large lambda expression libraries, which are rapidly and efficiently converted to stable plasmid libraries by mass excision. In SurfZAP, clones are expressed as fusions with amino acids 198-406 of the M13 minor coat protein (cpIII) and are displayed on the surface of filamentous phage. When produced with helper phage proteins, the fusion proteins are incorporated into the surface of phagemid particles. We demonstrate the utility of biopanning by isolating tetanus toxoid-binding mouse Fab clones from SurfZAP libraries. Approximately 10-100-fold enrichment of specific clones was observed after each panning round. The ability to create a large library of genotypes and screen the phenotypes by activity may be a potent methodology for basic research and drug discovery.

A bacteriophage lambda vector for the cloning and expression of immunoglobulin Fab fragments on the surface of filamentous phage.

We have combined the high cloning efficiency of the lambda 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 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 VH genes into ImmunoZAP 13, in vivo mass excision to convert the lambda 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.

Synthesis and characterization of a recombinant myohemerythrin protein encoded by a synthetic gene.

The antigenic epitopes of the myohemerythrin (MHr) molecule have been studied extensively. The critical amino acid residues responsible for its immune recognition have been identified by using synthetic peptides and the technique of epitope scanning. To assess the true relevance of these techniques for determining the molecular mechanism of antigenic recognition and immunogenicity, the results obtained with isolated peptides should be tested in the context of the folded protein. To this end, we have designed and constructed a synthetic MHr gene, in modular form, which will allow subsequent alterations of nucleotide sequence encoding epitopes of interest. We have produced the recombinant protein at high level, and have shown by several criteria that it possesses the chemical, physical and immunological properties of the native worm protein. Thus, we have developed a valuable system for detailed immunological studies of the structure and chemistry required for antibody binding to protein.

Expression of a heterodimeric Fab antibody protein in one cloning step.

A new method is presented for creating antibody expression libraries in Escherichia coli. Rather than perform two cloning steps to express the heavy and light chains of the antigen binding domain, we have used a fusion-PCR method to link the coding regions for heavy and light chain sequences in a single DNA molecule prior to vector ligation. This greatly simplifies the construction of antibody expression clones.

Ophthalmic manifestations of Allgrove syndrome: report of a case.

A male patient with the ocular manifestations of Allgrove or triple-A syndrome is described. The need for early diagnosis based on alacrima, anisocoria and optic atrophy of this potentially fatal condition is stressed.

Bacteriophage cloning and Escherichia coli expression of a human IgM Fab.

We have combined the molecular biology methods of the polymerase chain reaction and recombinant DNA cloning in bacteriophage lambda to express a human IgM Fab in Escherichia coli using genes derived from an Epstein-Barr virus transformed cell line. This method comprises three cDNA amplifications and a single cloning step, culminating in the stable overexpression of mammalian heterodimeric recombinant protein in a prokaryotic host.

Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library.

We have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and kappa light-chain variable and constant region domains, were inserted into modified bacteriophage lambda expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. We estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.

Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library.

Efficient generation of catalytic antibodies is uniquely dependent on the exact nature of the binding interactions in the antigen-antibody complex. Current methods for generation of monoclonal antibodies do not efficiently survey the immunological repertoire and, therefore, they limit the number of catalysts that can be obtained. We are exploring methods to clone and express the immunological repertoire in Escherichia coli. As the essential first step, we present here a method for the establishment of a highly diverse heavy chain variable region library. Consequently, it should now be possible to express and recombine the heavy and light chain variable region fragments to generate a large array of functional combining portions of the antibody molecule. This technology may provide an alternative to the hybridoma methodology for accessing the monoclonal antibody specificity of the immune system.