In vitro selection of enzymes by mRNA-Display

本项目的目标是使用不同的mRNA展示文库选择具有Michaelase和Aldol缩合活性的肽和蛋白质。mRNA展示是一种允许在mRNA或合成RNA文库与其编码的肽或蛋白质之间产生共价融合的技术。mRNA展示文库可以通过在其3'端携带嘌呤霉素（一种肽基受体抗生素）的合成mRNA的体外翻译来产生。所得接合分子的信息（核酸）和功能（肽）结构域之间的稳定连接允许基于其编码肽的性质从mRNA的复杂混合物中富集特定mRNA。我们将使用我们实验室中已经存在的mRNA展示文库，即完全随机化而无结构偏倚的文库，除了a-螺旋和b-折叠形成结构域的概率增加。另一个文库基于脂质运载蛋白支架，并且由支架的连接环结构域中的随机氨基酸组成。选择方案将基于对应于文库中每个序列的cDNA的化学衍生和标记。作为活性催化剂的那些肽序列将在cDNA上引入标签，使得其可以与非活性序列分离、扩增并用作随后选择循环的输入。这种策略应该允许从随机序列中选择寡肽的酶活性，而没有来自催化类似反应的天然酶的任何偏差。

The goal of this project is the selection of Peptides and Proteins with Michaelase and Aldol condensation activities using different mRNA-display libraries. mRNA-Display is a technique that allows to generate covalent fusions between an mRNA, or a library of synthetic RNAs, and the peptide or protein that it encodes. mRNA-display libraries can be generated by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3' end. The stable linkage between the informat ional (nucleic acid) and functional (peptide) domains of the resulting joint molecules allows a specific mRNA to be enriched from a complex mixture of mRNAs based on the properties of its encoded peptide. We will use mRNA-display libraries that are already available in our laboratory, namely a library that is completely randomised without structural bias, except for an increased probability of a-helical amd b-sheet-forming domains. The other library is based on the lipocalin scaffold and consists of random amino acids in the connecting loop domains of the scaffold. The selection scheme will be based on chemical derivatization and tagging of the cDNA that corresponds to each sequence in the library. Those peptide sequences that are active catalysts will introduce a tag onto the cDNA so that it can be separated from inactive sequences, amplified, and be used as the input for the subsequent selection cycle. This strategy should allow to select enzymatic activities of oligopeptides from random sequences without any bias from natural enzymes that catalyse similar reactions.