A simple vector system to improve performance and utilisation of recombinant antibodies

Abstract

Background: Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression.

Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain

“…In addition to affinity-based approaches, a “competitive elution” selection was performed in parallel in which MET-bound phage populations were incubated with high (micromolar) concentrations of the ligand HGF/SF in an effort to elute HGF/SF-competitive antibodies. Selected populations were cloned into a bacterial expression vector 35 and individual clones were screened directly for functional activity using a colony scatter assay. Hits emerging from this screen were assessed in a more quantitative cell migration assay, based on migration of fluorescently labeled SKOV3 human ovarian cancer cells using a Boyden chamber.…”

“…Biopanning with the chain-shuffled library (10 9 clones) was performed with biotinylated MET928 in solution and streptavidin-coated dynabeads. Phage pools were cloned into expression vector 35 and small-scale expressions performed in BL21 (DE3) bacteria in 96-well format. Approximately 960 supernatants were screened directly for inhibition of HGF/SF-induced scatter of BxPC-3 human pancreatic cancer cells.…”

BLM helicase facilitates telomere replication during leading strand synthesis of telomeres

“…The single-chain antibody BG4 was isolated from the Sanger phage-display library (2.3 × 10 10 single-chain antibody clones) by selection against a panel of intramolecular G4 structures ( Biffi et al, 2013 ). The BG4 sequence was then cloned into the pSANG10 expression plasmid ( Martin et al, 2006 ) to generate pSANG10-BG4, which expresses the BG4 antibody along with C-terminal hexa-histidine and 3×FLAG epitope tags. The BG4 antibody was expressed from pSANG10-BG4 in bacterial cultures grown overnight in auto-induction medium as described previously ( Martin et al, 2006 ).…”

“…The BG4 sequence was then cloned into the pSANG10 expression plasmid ( Martin et al, 2006 ) to generate pSANG10-BG4, which expresses the BG4 antibody along with C-terminal hexa-histidine and 3×FLAG epitope tags. The BG4 antibody was expressed from pSANG10-BG4 in bacterial cultures grown overnight in auto-induction medium as described previously ( Martin et al, 2006 ). After overnight culturing, the cells were pelleted and periplasmic extracts were prepared by resuspending pellets in ice-cold TES buffer (50 mM Tris, pH 8, 1 mM EDTA, and 20% sucrose), placing them on ice for 10 min followed by the addition of an equal volume of 0.2× TES buffer, and keeping them on ice for an additional 15 min.…”

Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers

“…For kinetic studies, cell binding and in vivo experiments, scFv clones were produced by inoculating shake flasks containing 50 - 100 ml 2YT media (supplemented with 50 μg/ml kanamycin) with the E. coli strain BL21(DE3) harboring the individual scFv clones in the expression vector pSANG10-3F. When cultures reached OD600 = 0.8 - 1, expression was induced by addition of 1 mM isopropyl-beta-D-1-thiogalactopyranoside (IPTG) and, following over night incubation at +30°C, scFv were recovered from the periplasm as previously described [ 44 ]. Periplasmic fractions were further affinity purified (Nickel affinity gel, Sigma-Aldrich, #P6611).…”