Saturation mutagenesis is a powerful tool in modern protein engineering. This can allow the analysis of potential new properties thus allowing key residues within a protein to be targeted and randomised. However, the creation of large libraries using conventional saturation mutagenesis with degenerate codons (NNN or NNK) has inherent redundancy and disparities in residue representation. In this we describe the combination of ProxiMAX randomisation and CIS display for the use of generating novel peptides. Unlike other methods ProxiMAX randomisation does not require any intricate chemistry but simply utilises synthetic DNA and molecular biology techniques. Designed ‘MAX’ oligonucleotides were ligated, amplified and digested in an iterative cycle. Results show that randomised ‘MAX’ codons can be added sequentially to the base sequence creating a series of randomised non-degenerate codons that can subsequently be inserted into a gene. CIS display (Isogencia, UK) is an in vitro DNA based screening method that creates a genotype to phenotype link between a peptide and the nucleic acid that encodes it. The use of straight forward in vitro transcription/translation and other molecular biology techniques permits ease of use along with flexibility making it a potent screening technique. Using ProxiMAX randomisation in combination with CIS display, the aim is to produce randomised anti-nerve growth factor (NGF) and calcitonin gene-related (CGRP) peptides to demonstrate the high-throughput nature of this combination.
|Number of pages||1|
|Publication status||Published - Jul 2013|
|Event||Protein engineering: new approaches and applications - University of Chester, Chester, United Kingdom|
Duration: 10 Apr 2013 → 12 Apr 2013
|Conference||Protein engineering: new approaches and applications|
|Period||10/04/13 → 12/04/13|
|Other||A joint Biochemical Society / Protein Society conference. Protein engineering has proved pivotal to our molecular understanding and adaption of proteins by allowing us to interact and manipulate the very coding sequence that underlies folding, structure and function.|
Protein engineering is currently undergoing a renaissance with the advent of new technological approaches to address some of major emerging areas in the life sciences, including facilitating the interface with both chemistry and physics. Protein engineering will prove central to areas ranging from synthetic biology to bioprocessing to nanotechnology.
In recent years the traditional idea of simple site directed mutagenesis has been superseded through the developed of new approaches and technologies that have enabled the field of protein engineering to flourish. These new developments that include advanced computational design, development of new and useful biocatalysts, integration of functional biological parts with fabricated devices and construction of next generation biopharmaceuticals. Protein engineering also continues to provide valuable and fundamental understanding of natural protein construction and function that in turn will inevitably improve our ability to generate the next generation of novel proteins.
This meeting will assemble a diverse group of world-leading protein engineers who will highlight the recent advances in our current understanding of how proteins can be tractably manipulated towards an intended function either through rational design or directed evolution.
Poole, A. J., Ullman, C. G., & Hine, A. V. (2013). The integration of ProxiMAX randomisation with CIS display for the high-throughput production of novel peptides. P049. Poster session presented at Protein engineering: new approaches and applications, Chester, United Kingdom.