Directed Evolution of Improved Orthogonal Aminoacyl-tRNASynthetases Through Phage Assisted Continuous Evolution (PACE)

通过噬菌体辅助连续进化 (PACE) 改进正交氨酰基-tRNA 合成酶的定向进化

• 批准号: 8833708
• 负责人: David Bryson
• 金额: $ 5.42万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8833708
• 关键词: Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Animals; Bacteriophages; Binding; Biological Process; Cells; Charge; Chemical Structure; Chemicals; Codon Nucleotides; Custom; Ensure; Enzymes; Epigenetic Process; Escherichia coli; Evolution; Genetic Code; Goals; Haptens; Hour; Intervention; Investments; Laboratories; Length; Libraries; Life; Lysine; Mammals; Methods; Mutation; Performance; Phenylalanine; Post-Translational Protein Processing; Proteins; Research; Research Personnel; Role; Site; Specific qualifier value; Specificity; Substrate Specificity; Techniques; Testing; Therapeutic; Time; Transfer RNA; Translations; Variant; Work; directed evolution; histone methylation; immunogenic; improved; interest; prevent; public health relevance; research study; stem; success; synthetic biology; tool

 DESCRIPTION (provided by applicant): Orthogonal translation of modified proteins featuring genetically encoded non-natural amino acids (nnAA) such as photocrosslinkers, photocaged groups, biophysical probes, posttranslational modifications, and proximity enhanced bioreactive groups has become an important tool for the study of biological processes and has achieved a long-standing goal of synthetic biology. A critical requirement for site-specific incorporation of nnAA into a custom protein is the ability to selectively catalyze aminoacylation of an orthogonal tRNA with the specified amino acid. Therefore, directed evolution of a new orthogonal aminoacyl-tRNA synthetase (aaRS) is required for each new nnAA that is added to the growing amino acid repertoire. While repeated cycles of positive and negative selection have allowed nonnatural aminoacylation activity to be evolved successfully, these efforts typically utilize only 3-5 total rounds of evolution due to the difficulty and time investment that is required to carry ot each round of traditional laboratory evolution. Unfortunately, these efforts result in evolved aaRS enzymes with relatively modest selectivity and greatly reduced activity compared to wild-type enzymes. This proposed work would test the hypothesis that selecting for new aaRS variants through massively increased total rounds of evolution will allow highly selective aaRSs to be evolved with wild-type-like activity. Phage-assisted continuous evolution (PACE) enables proteins to be evolved in the laboratory at a rate of up to ~40 rounds of evolution every 24 hours with minimal researcher intervention. In this proposed research, positive and negative selections using PACE will be used in an attempt to rapidly evolve enhanced orthogonal aaRSs capable of selective aminoacylation with desired nnAAs. Specific Aim 1 seeks to improve the selectivity of the p-nitrophenylalanyl-tRNA synthetase, which has previously been used to site-selectively incorporate a proximity probe into a designer protein and has also successfully been used as an immunogenic hapten in a protein. Specific Aim 2 will attempt to improve upon the inherently low activity of the pyrrolysyl-tRNA synthetase (PylRS), which has permitted genetic code expansion in cells and animals including mammals. Finally, Specific Aim 3 will seek to evolve a variant of the PylRS that is capable of selective aminoacylation with unprotected N6,N6-dimethyl-L-lysine in the presence of cellularly abundant L-lysine, which would have great utility in studies of posttranslational modifications such as histone methylation. If successful, this research will establish a new platform for rapidly evolving enhanced orthogonal aaRS enzymes to greatly expand their use in synthetic biology and therapeutic applications.

 描述（由申请人提供）：以遗传编码的非天然氨基酸（nnAA）为特征的修饰蛋白质（如光交联剂、光笼基团、生物物理探针、翻译后修饰和邻近增强生物活性基团）的正交翻译已成为研究生物过程的重要工具，并实现了合成生物学的长期目标。将nnAA位点特异性掺入定制蛋白质的关键要求是选择性催化正交tRNA与指定氨基酸的氨酰化的能力。因此，一个新的正交氨酰-tRNA合成酶（阿尔斯）的定向进化是需要每个新的nnAA被添加到不断增长的氨基酸库。虽然正选择和负选择的重复循环已经允许成功地进化非天然氨酰化活性，但是这些努力通常仅利用 3-5由于进行每一轮传统实验室进化所需的难度和时间投入，不幸的是，这些努力导致进化的阿尔斯酶与野生型酶相比具有相对适度的选择性和大大降低的活性。这项拟议的工作将测试这样的假设，即通过大量增加的总进化轮次来选择新阿尔斯变体将允许高度选择性的aaRS进化为具有野生型样活性。 噬菌体辅助连续进化（PACE）使蛋白质能够在实验室中以每24小时约40轮进化的速度进化，而研究人员的干预最少。在这项拟议的研究中，使用PACE的阳性和阴性选择将用于尝试快速进化能够与所需nnAA选择性氨酰化的增强的正交aaRS。具体目标1寻求提高对硝基苯丙氨酰-tRNA合成酶的选择性，该合成酶先前已用于将邻近探针位点选择性地掺入设计蛋白中，并且还已成功地用作蛋白质中的免疫原性半抗原。具体目标2将试图改善吡咯赖氨酰-tRNA合成酶（PylRS）固有的低活性，其允许在细胞和动物（包括哺乳动物）中进行遗传密码扩增。最后，具体目标3将寻求进化PylRS的变体，其能够在细胞丰富的L-赖氨酸存在下与未保护的N6，N6-二甲基-L-赖氨酸选择性氨酰化，这将在翻译后修饰如组蛋白甲基化的研究中具有很大的实用性。如果成功，这项研究将建立一个新的平台，快速发展增强正交阿尔斯酶，大大扩大其在合成生物学和治疗应用中的用途。