A revised strategy for cloning antibody gene fragments in bacteria

• Published in: Gene Vol. 128; no. 1; pp. 111 - 117
• Main Authors: Posner, B., Lee, I., Itoh, T., Pyati, J., Graff, R., Thorton, G.B., La Polla b, R., Benkovic, S.J.
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Lausanne Elsevier B.V 01.01.1993; Amsterdam Elsevier; New York, NY
• Subjects: affinity-tail sequence; Amino Acid Sequence; Artificial operon; Bacteriophage lambda - genetics; Base Sequence; Biological and medical sciences; Biotechnology; Cloning, Molecular - methods; coliphage T3 promoter; combinatorial library; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene Library; Genetic engineering; Genetic technics; Immunoglobulin Fab Fragments - biosynthesis; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - isolation & purification; Immunoglobulin Heavy Chains - biosynthesis; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Light Chains - biosynthesis; Immunoglobulin Light Chains - genetics; Methods. Procedures. Technologies; Molecular Sequence Data; monocistronic expression; Oligodeoxyribonucleotides; Oligopeptides - biosynthesis; plaque-lift assay; Plasmids; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; Restriction Mapping; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Ab; monocistronic expression; EDC; nt; NPN; bp; Ap; Tc; tsp; BSA; kb; NHS; plaque-lift assay; wt; Fd; combinatorial library; T3; aa; MOP3; NBT; RBS; SDS; bla (or Ap r ); TBS; Fab; Artificial operon; ori; PAGE; mAb; lacP; p; r; lacO; coliphage T3 promoter; ε; Bla; LB; BCIP; IPTG; affinity-tail sequence; κ; MCS; Ig; PCR; ELISA; Heterologous system; Antibody; Transcription promoter; Rodentia; Siphoviridae; Coliphage; Fab-Fragment; Gene expression; Virus; Vertebrata; Mammalia; Mouse; Bacteria; Phage lambda; Molecular cloning; Vector; Phage
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(93)90161-U

The ability to clone and overexpress genes encoding mouse Fab (antigen-binding fragment) proteins in bacteria led to the development of a methodology which has the potential to replace traditional hybridoma technology [Huse et al., Science 246 (1989) 1275–1281]; however, several observations have suggested that clones with desirable chemical properties may be missed in immunoscreens of large combinatorial libraries due to low levels of functional Ab protein. To increase the efficiency of cloning and characterization of Ab gene fragments, we have reconsidered several features of the original cloning vehicles. These studies show that at the present time a unique expression system cannot adequately accommodate the requirements of plaque-lift immunoassays for clonal selection and biochemical assays for further characterization in vitro. A monocistronic arrangement of heavy- and light-chain-encoding genes using two lacP promoters produces sufficient amounts of functional Ab protein for clonal selection from phage λ libraries and minimizes interference with the lytic cycle of recombinant vectors. In liquid culture, a strong coliphage promoter and a relatively abundant RNA polymerase can be used to produce quantities of Ab protein sufficient for further characterization in vitro. A rapid purification protocol obviates the need for fusing heavy-chain protein to a decapeptide sequence, an affinity-tail sequence which slows the folding and assembly of the Ig heterodimer. These results have been used to formulate a new strategy for cloning and characterization of Ab gene fragments in bacteria.