A holistic approach to rapid protein engineering: the synergistic combination of optimised protein library generation and screening.

This proposal aims to develop and combine 'MAX' and 'ProxiMAX' randomisation technologies with Isogenica's proprietary CIS screening technology, to generate a seamless platform technology that addresses all key issues of protein engineering simultaneously. Both 'MAX' randomisation technologies were developed at Aston University with BBSRC support (grants B14245 and BB/D525756/1 respectively) and subsequent in-house funding. CIS screening, developed by Isogenica, represents the state-of-the-art in terms of screening engineered protein libraries. Current approaches to high-throughput protein engineering optimise either mutagenesis, or screening. Few optimise both (and those that do, rely on complex, expensive, chemical synthesis of libraries). This project will represent the first combination of simple, optimised mutagenesis AND screening, where library size is minimised, whilst screening capacity is maximised, thus enabling diversity greater than previously achievable, from simple, in vitro-based technology. Moreover, rather than utilising model proteins, the project will employ peptide targets for development of current academic/commercial relevance. Conceptually, a protein/peptide is a string of amino acids folded into a 3D structure that defines activity. If one or more amino acids are changed, protein activity may be unchanged, abolished or altered. The latter is protein engineering's goal: to generate synthetic proteins with novel or enhanced activities. Commercially, protein engineering is performed in high-throughput, either by saturation mutagenesis (where structural data is available); gene shuffling (where information is lacking) or by a combination of the two approaches. Whichever is employed, a protein library results that is encoded by variants of just one gene. Within saturation mutagenesis, that variation is limited to specific codon(s), replaced with randomised codon(s) such as NNN or NNG/T (N is any nucleotide). Once expressed, the library is screened to find the 'best' protein with the new, required activity. Unfortunately, saturation mutagenesis has drawbacks (associated with genetic code degeneracy) including high wastage ratios and unequal protein concentrations resulting in protein libraries that are likely to compromise screening processes. 'MAX' and 'ProxiMAX' randomisation are technologies that each address the pitfalls of saturation mutagenesis, enabling combinatorial protein engineering that was previously impossible, except by using highly-specialised chemistry. By eliminating genetic code degeneracy from the libraries, they deliver small libraries that should produce the 'best' protein every time, for vastly-reduced screening costs. 'MAX' was developed to engineer proteins where the key residues are sequentially separated (e.g. the residues within the active site of an enzyme), whereas 'ProxiMAX' is used to randomise contiguous amino acids. By removing the need for cloning, CIS screening eliminates toxicity effects and cloning bias from protein libraries and increases the maximum achievable library size by 4-5 orders of magnitude. Genes are expressed in vitro, as fusions to RepA protein. The DNA expression cassettes also contain CIS and ori sites which together, cause each newly-expressed RepA fusion to bind to its encoding DNA. This DNA (successful proteins) is amplified by PCR. Only after several cycles of 'panning' are the few resulting cassettes cloned and their proteins identified / over-expressed. In integrating these two broadly-compatible technologies, we aim to engineer both peptide modulators of Nerve Growth Factor (NGF - of particular interest to Isogenica); and peptide variants of Calcitonin Gene Related Peptide (CGRP - of particular interest to Aston University). Key research challenges will include the introduction of programmed bias into 'MAX'/'ProxiMAX' randomisation, development of technology compatibility and engineering of specific peptide products.