RESUMO
Affinity reagents, such as antibodies, are needed to study protein expression patterns, sub-cellular localization, and post-translational modifications in complex mixtures and tissues. Phage Emulsion, Secretion, and Capture (ESCape) is a novel micro-emulsion technology that utilizes water-in-oil (W/O) emulsions for the identification and isolation of cells secreting phage particles that display desirable antibodies. Using this method, a large library of antibody-displaying phage will bind to beads in individual compartments. Rather than using biopanning on a large mixed population, phage micro-emulsion technology allows us to individually query clonal populations of amplified phage against the antigen. The use of emulsions to generate microdroplets has the promise of accelerating phage selection experiments by permitting fine discrimination of kinetic parameters for binding to targets. In this study, we demonstrate the ability of phage micro-emulsion technology to distinguish two scFvs with a 300-fold difference in binding affinities (100nM and 300pM, respectively). In addition, we describe the application of phage micro-emulsion technology for the selection of scFvs that are resistant to elevated temperatures.
Assuntos
Técnicas de Visualização da Superfície Celular , Evolução Molecular Direcionada , Anticorpos de Cadeia Única/genética , Afinidade de Anticorpos , Bacteriófago M13/genética , Emulsões , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Humanos , Cinética , Mutagênese , Biblioteca de Peptídeos , Reação em Cadeia da Polimerase , Ligação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/químicaRESUMO
Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>10(20)), the practical diversity that can be achieved by transformation of Escherichia coli is limited to about 10(10). To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target.
Assuntos
Biblioteca de Peptídeos , Anticorpos de Cadeia Única/genética , Sequência de Aminoácidos , Diversidade de Anticorpos , Biotecnologia , Regiões Determinantes de Complementaridade/genética , Escherichia coli/genética , Ensaios de Triagem em Larga Escala , Humanos , Anticorpos de Cadeia Única/biossínteseRESUMO
The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones.
Assuntos
Metilases de Modificação do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Mutagênese Sítio-Dirigida/métodos , Clonagem Molecular , Metilases de Modificação do DNA/biossíntese , DNA Recombinante/genética , DNA Recombinante/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/biossíntese , Biblioteca Gênica , Variação Genética , Vetores Genéticos/genéticaRESUMO
Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes.