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T Clackson

Publications and source records attributed to T Clackson.

22 records · Page 2Linked to original sources
PubMed1994

In vitro selection from protein and peptide libraries.

In vitro selection from molecular libraries has rapidly come of age as a protein-engineering tool. Dramatic increases in protein affinity can be engineered using phage-display libraries, and specific antibodies can be selected directly from a single 'naïve' library of their genes. Repertoires of small molecules are a potentially valuable resource for drug discovery. Libraries of linear peptides provide ligands for proteins that recognize continuous epitopes, and low-affinity mimics of some small molecules, but generally do not contain mimics of large molecular interfaces. Switching to constrained peptide formats, and deploying more diverse, non-peptide chemical libraries, may bring greater success.

Animals↗
PubMed1991

Making antibody fragments using phage display libraries.

To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.

Amino Acid Sequence↗
PubMed1989

'Sticky feet'-directed mutagenesis and its application to swapping antibody domains.

We describe a novel technique for precisely cutting and pasting two DNA sequences without using restriction sites. The method is based on site-directed mutagenesis and uses a long primer, generated by the polymerase chain reaction (PCR), to transfer large segments of DNA into a single-stranded template. The primer anneals to the template by virtue of 'sticky feet' sequences (complementary to the template) which are introduced at the ends of the primer by the PCR. Yields of desired recombinants were high (approximately 36%) and the transplanted sequences (approximately 400bp) free of errors. We have used this technique to swap CH2 domains between two mouse antibodies, and find that this domain can carry features critical for triggering complement lysis, in addition to the C1q binding motif.

Animals↗