DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

111. Developing General Methods to Select Phage Antibodies Against Gene Products

Peter Pavlik¹, Robert Siegal¹, Daniele Sblattero², Vittorio Verzillo^1,2, Roberto Marzari³, Jianlong Lou², Jim Marks⁴, and Andrew Bradbury^1,2

¹Los Alamos National Laboratory, Los Alamos, NM 87545; ²SISSA, Trieste, Italy; ³University of Trieste, Trieste, Italy; and ⁴University of California San Francisco, San Francisco, CA

amb@lanl.gov

Phage display offers the possibility of selecting polypeptides (and the genes which encode them) from libraries of 1e10 or more different polypeptides on the basis of their abilities to bind target proteins and subdomains. This diversity far surpasses the estimated number of total genes in the human genome. The application of this technology to the Human Genome Project will powerfully accomplish a central goal: the derivation of ligands that recognize protein products of all human genes, such ligands being either antibodies, or protein fragments.

Where the recognition ligands derived from this relatively new technology are antibody binding regions (single chain Fv) they can be employed in the same way as traditional antibodies. As such, they can play essential roles in assigning gene function, including the characterization of spatiotemporal patterns of protein expression and the elucidation of protein-protein interactions. Where the recognition ligands are protein fragments, they can be considered to be potential protein-interaction partners for the immobilized polypeptide and so a starting point for further biochemical studies.

This project has concentrated on trying to find a general way to isolate antibodies against gene products, preferably starting from gene sequence and using peptides to avoid the need for cloning and expression, although high throughput methods to select against recombinant products have also been developed.

Selection of antibodies against recombinant proteins has been reduced to the microtitre format. A comparison of the antibodies selected using this protocol with the standard selection procedure shows that the antibodies selected are on the whole different, although there is some overlap. Should gene products be available this is a very efficient way to select antibodies in a high throughput format.

In addition, selection on peptide surrogates of gene products has also been attempted. 192 scanning peptides corresponding to overlapping parts of four different proteins have been synthesised on microtitre pins and used to select phage antibodies. Some of the selected antibodies are able to recognise the full length protein. An analysis of the peptides which select antibodies recognising the full length protein has allowed us to develop an algorithm to predict which peptides are more likely to select useful proteins.