Directed Evolution of Formylglycine-generating Enzyme to Build an Optimal Platfor

甲酰甘氨酸生成酶的定向进化构建最佳平台

• 批准号: 8709882
• 负责人: David Ian Rabuka
• 金额: $ 13.63万
• 依托单位: REDWOOD BIOSCIENCE, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-17 至 2015-03-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8709882
• 关键词: Adhesions; Adverse effects; Aerobic; Agglutinins; Aldehydes; Amino Acid Sequence; Biological; Biological Assay; Biological Response Modifier Therapy; Businesses; Catalysis; Cell Surface Proteins; Cell surface; Cells; Chemicals; Complex; Consensus Sequence; Cysteine; Development; Drug Kinetics; Engineering; Enzymes; Flow Cytometry; Fluorescence-Activated Cell Sorting; Fluorescent Dyes; Generations; Hand; Human; Immune response; Immunofluorescence Immunologic; In Vitro; Intellectual Property; Label; Lead; Libraries; Methods; Modification; Monitor; Peptide Sequence Determination; Peptides; Phase; Plasmids; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Recombinant Proteins; Recombinants; Redwood; Research; Saccharomyces cerevisiae; Serum Albumin; Site; Specificity; Surface; System; Technology; Testing; Therapeutic; Treatment Efficacy; Variant; Yeasts; base; commercial application; design; directed evolution; enzyme activity; enzyme substrate; expression vector; flexibility; formylglycine; in vivo; interest; mutant; novel; novel therapeutics; overexpression; pressure; programs; public health relevance; synthetic peptide; therapeutic protein

DESCRIPTION (provided by applicant): The formylglycine generating-enzyme (FGE) recognizes and acts specifically on a pentapeptide (CXPXR), oxidizing the cysteine to an unusual aldehyde bearing formylglycine residue (fGly). This conversion to fGly is possible when the sequence is inserted into recombinant proteins. The small FGE target sequence, termed the "aldehyde tag", anchors the Redwood Bioscience technology that can generate homogenous, enhanced biotherapeutics. This platform provides the site specificity and chemical flexibility needed to generate useful post translational modifications on virtually any protein. This ability o affix novel chemical functionalities to a biological entity of interest is an immensely powerful platform for the development and optimization of new therapeutics. As applications of this technology are explored it will be advantageous to have flexibility in the consensus sequence recognized by FGE, so as to permit the modification of as many protein targets as possible, ideally on sites that exist already in their endogenous sequences. A panel of FGE enzyme/tag pairs would be an incredibly powerful platform and generate valuable intellectual property. The new tag sequences will be designed to minimize perturbation of the protein sequence and maximize FGE specific activity, thereby enabling optimized conjugation. This new tag technology will generate bioconjugates with minimal antigenicity and enhanced pharmacokinetics. Towards this end, Redwood Bioscience will embark on a research program using a directed evolution approach with yeast cell surface protein display to generate FGE variants that recognize unique and novel substrates.

描述(由申请人提供)：甲酰甘氨酸生成酶(FGE)识别并特异性地作用于五肽(CXPXR)，将半胱氨酸氧化成含有甲酰甘氨酸残基(FGly)的不寻常的醛。当序列插入到重组蛋白中时，这种转化为fGly是可能的。被称为“醛标签”的小FGE靶序列锚定了红杉生物科学技术，该技术可以产生同质的、增强的生物治疗药物。该平台提供了在几乎任何蛋白质上产生有用的翻译后修饰所需的位点特异性和化学灵活性。这种将新的化学功能附加到感兴趣的生物实体上的能力是开发和优化新疗法的强大平台。随着这项技术的应用被探索，在FGE识别的共识序列中具有灵活性将是有利的，以便允许对尽可能多的蛋白质靶标进行修饰，理想的是在其内源序列中已经存在的位点上进行修饰。一组FGE酶/标签对将是一个令人难以置信的强大平台，并产生宝贵的知识产权。新的标签序列将被设计成最大限度地减少蛋白质序列的扰动，并最大限度地提高FGE的比活性，从而实现优化的结合。这项新的TAG技术将产生抗原性最低的生物结合物和增强的药代动力学。为此，红木生物科学公司将启动一项研究计划，利用酵母细胞表面蛋白展示的定向进化方法来产生识别独特和新底物的FGE变体。