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

该提案旨在开发和结合“MAX”和“ProxiMAX”随机化技术与Isogenica专有的CIS筛选技术，以产生一个无缝的平台技术，同时解决蛋白质工程的所有关键问题。这两种“MAX”随机化技术都是在阿斯顿大学的BBSRC支持下开发的（分别获得了B14245和BB/D525756/1的资助）以及随后的内部资助。CIS筛选，由Isogenica开发，代表了最先进的筛选工程蛋白库。目前的高通量蛋白质工程方法要么优化诱变，要么优化筛选。很少有人能同时优化这两方面（即使是这样做的，也依赖于复杂、昂贵的化学合成库）。该项目将代表简单的、优化的诱变和筛选的第一次结合，其中文库大小最小化，同时筛选能力最大化，从而使多样性比以前通过简单的体外技术实现的更大。此外，该项目将采用肽靶标来开发当前的学术/商业相关性，而不是利用模型蛋白。从概念上讲，蛋白质/肽是一串折叠成3D结构的氨基酸，它定义了活性。如果一种或多种氨基酸发生改变，蛋白质活性可能保持不变、消失或改变。后者是蛋白质工程的目标：生成具有新颖或增强活性的合成蛋白质。商业上，蛋白质工程以高通量进行，要么通过饱和诱变（在结构数据可用的情况下）；基因洗牌（缺乏信息）或两种方法的结合。无论采用哪一种方法，都会产生一个仅由一个基因变体编码的蛋白质文库。在饱和诱变中，变异仅限于特定的密码子，取而代之的是随机密码子，如NNN或NNG/T （N是任何核苷酸）。一旦表达，文库将被筛选，以找到具有新所需活性的“最佳”蛋白质。不幸的是，饱和诱变有缺点（与遗传密码退化有关），包括高流失率和不均匀的蛋白质浓度，导致蛋白质文库可能损害筛选过程。“MAX”和“ProxiMAX”随机化技术都解决了饱和诱变的缺陷，实现了以前不可能的组合蛋白质工程，除非使用高度专业化的化学。通过消除文库中的遗传密码退化，他们提供了每次都能产生“最佳”蛋白质的小文库，从而大大降低了筛选成本。“MAX”被开发用于工程蛋白质，其中关键残基按顺序分离（例如酶活性位点内的残基），而“ProxiMAX”用于随机排列连续的氨基酸。通过消除克隆的需要，CIS筛选消除了蛋白质文库的毒性效应和克隆偏见，并将可实现的最大文库大小增加了4-5个数量级。基因在体外以与RepA蛋白融合的方式表达。DNA表达盒还包含CIS和ori位点，它们共同导致每个新表达的RepA融合与其编码DNA结合。这种DNA（成功的蛋白质）通过PCR扩增。只有经过几个周期的“筛选”后，才能克隆出少量的结果磁带，并鉴定出/过表达它们的蛋白质。整合这两种广泛兼容的技术，我们的目标是设计神经生长因子（NGF -特别感兴趣的Isogenica）的肽调节剂；以及降钙素基因相关肽（CGRP -阿斯顿大学特别感兴趣）的肽变体。关键的研究挑战将包括在“MAX”/“ProxiMAX”随机化中引入程序化偏差，开发技术兼容性和特定肽产品的工程。

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.