Nondegenerate Saturation Mutagenesis: Library Construction and Analysis via MAX and ProxiMAX Randomization.

Protein engineering can enhance desirable features and improve performance outside of the natural context. Several strategies have been adopted over the years for gene diversification, and engineering of modular proteins in particular is most effective when a high-throughput, library-based approach is employed. Nondegenerate saturation mutagenesis plays a dynamic role in engineering proteins by targeting multiple codons to generate massively diverse gene libraries.

A C-terminal cysteine residue is required for peptide-based inhibition of the NGF/TrkA interaction at nM concentrations: implications for peptide-based analgesics.

Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads.

Exploiting Overlapping Advantages of In Vitro and In Cellulo Selection Systems to Isolate a Novel High-Affinity cJun Antagonist.

We have combined two peptide library-screening systems, exploiting the benefits offered by both to select novel antagonistic agents of cJun. CIS display is an in vitro cell-free system that allows very large libraries (≤10) to be interrogated. However, affinity-based screening conditions can poorly reflect those relevant to therapeutic application, particularly for difficult intracellular targets, and can lead to false positives.

ProxiMAX randomization: a new technology for non-degenerate saturation mutagenesis of contiguous codons.

Back in 2003, we published 'MAX' randomization, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. 'MAX' randomization saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an α-helix, as in zinc-finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple contiguous codons in a non-degenerate manner.

In vitro methods for peptide display and their applications.

The presentation of recombinant peptide libraries linked to their coding sequence can be referred to as 'peptide display'. Phage display is the most widely practiced peptide display technology but more recent alternatives such as CIS display, ribosome display and mRNA display offer advantages over phage for speed, library size and the display of unnatural amino acids. These have provided researchers with tools to address some of the failings of peptides such as their low affinity, low stability and inability to cross biological membranes.

High affinity nucleic acid aptamers for streptavidin incorporated into bi-specific capture ligands.

We have isolated 2'-Fluoro-substituted RNA aptamers that bind to streptavidin (SA) with an affinity around 7 +/- 1.8 nM, comparable with that of recently described peptide aptamers. Binding to SA was not prevented by prior saturation with biotin, enabling nucleic acid aptamers to form useful ternary complexes.