Journal: Nucleic Acids Research

Article Title: Covalent antibody display—an in vitro antibody-DNA library selection system

doi: 10.1093/nar/gni010

Figure Lengend Snippet: ( a ) The endonuclease P2A (761 residues, 86.3 kDa) makes a single-stranded site-specific nick at Ori of replication (…CCT CGG, *), located inside its own gene at position 1860, and becomes covalently attached (via Y454) to the 5′ phosphate of its own DNA ( – ). ( b ) CAD is the exploitation of P2A to select antibodies or antibody fragments genetically fused to it. In prokaryotes, the transcription and translation are coupled, and the start of translation normally takes place before the transcription is finished . The figure is a model of the complex formation among DNA, RNA polymerase, ribosomes, mRNA and nascent P2A–scFv fusion proteins (colours match the gene products). The P2A protein part of the P2A–scFv fusion protein indirectly attaches the genotype of the scFv covalently to its phenotype. (L = linker). ( c ) Selection cycle for CAD. An scFv repertoire is assembled with the P2A gene using PCR methods and fresh P2A and tac-promoter (1). Protein–DNA complexes are being produced in an in vitro E.coli S30 coupled transcription–translation mixture (2) and selected on the immobilized target (3 and 4). Retained members are eluted and DNA for the next cycle is prepared using PCR (5). Figures are not drawn to scale.

Article Snippet: For a 50 μl reaction in an Eppendorf tube, the following components were assembled on ice: S30 2.5× reaction buffer (20 μl), E.coli S30 extract (15 μl), ∼1 mM l -methionine (Sigma), 1 μM protein disulfide isomerase (PDI; Sigma), 40 U RNasin (Promega), nuclease-free water, 0.1% BSA and DNA (1–2.5 μg).

Techniques: Selection, Produced, In Vitro

Journal: Nucleic Acids Research

Article Title: Covalent antibody display—an in vitro antibody-DNA library selection system

doi: 10.1093/nar/gni010

Figure Lengend Snippet: ( a ) Western/ECL identification of P2A-HA (lane 1) and P2A-anti-DOB3-c-Myc (lane 2) proteins expressed in a coupled transcription-translation mixture (Promega E.coli S30 extract for linear templates). The proteins were separated on 12.5% criterion polyacrylamide gel (Bio-Rad), blotted onto nitrocellulose membrane and detected by anti- c -Myc/anti-HA and rabbit anti-mouse IgG-HRP/ECL. ( b ) Agarose gel of rescue PCR of DNA from anti-phOx-P2A (left panel) or P2A-anti-phOx (right panel) protein–DNA complexes selected onto target T (phOx–BSA-coated Dynabeads) or negative control NC (BSA-coated Dynabeads). The protein–DNA complexes were generated in E.coli S30 lysate added 1 μM PDI and 1 mM DTT. They were eluted with proteinase K digestion, alcohol precipitated and detected by PCR. ( c ) Agarose gel of rescue PCR of DNA from scFv(E7)–P2A protein–DNA complexes selected on N.meningitidis outer membrane vesicles (T) or superblock (Pierce, NC). The effect of RNase (Promega) treating samples immediately after transcription–translation (at 30°C, 10 U) or adding 0.1% BSA during transcription and translation. The DNA bands from no additions/post-treatments are also shown (0).

Article Snippet: For a 50 μl reaction in an Eppendorf tube, the following components were assembled on ice: S30 2.5× reaction buffer (20 μl), E.coli S30 extract (15 μl), ∼1 mM l -methionine (Sigma), 1 μM protein disulfide isomerase (PDI; Sigma), 40 U RNasin (Promega), nuclease-free water, 0.1% BSA and DNA (1–2.5 μg).

Techniques: Western Blot, Agarose Gel Electrophoresis, Negative Control, Generated