Svetlana P. Ikonomova, Bo Yan, Zhiyi Sun, Rachel B. Lyon, Kelly M. Zatopek, John P. Marino, Zvi Kelman
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To fill this gap, a human protein GID4 was engineered to create a NAAB for N-terminal proline (Nt-Pro). While native GID4 binds Nt-Pro, its binding is weak (µmol/L) and greatly influenced by the identity of residues following the Nt-Pro. Through directed evolution, yeast-surface display, and fluorescence-activated cell sorting, we identified sequence variants of GID4 with increased binding response to Nt-Pro. Moreover, variants with an A252V mutation showed a reduced influence from residues in the second and third positions of the target peptide when binding to Nt-Pro. The workflow outlined here is shown to be a viable strategy for engineering NAABs, even when starting from native Nt-binding proteins whose binding is strongly impacted by the identity of residues following Nt-amino acid.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"7 1","pages":""},"PeriodicalIF":3.5000,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Engineering GID4 for use as an N-terminal proline binder via directed evolution\",\"authors\":\"Svetlana P. Ikonomova, Bo Yan, Zhiyi Sun, Rachel B. Lyon, Kelly M. Zatopek, John P. Marino, Zvi Kelman\",\"doi\":\"10.1002/bit.28868\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Nucleic acid sequencing technologies have gone through extraordinary advancements in the past several decades, significantly increasing throughput while reducing cost. 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引用次数: 0
摘要
过去几十年来,核酸测序技术取得了长足的进步,在降低成本的同时大幅提高了产量。为了在蛋白质组学方面取得类似的进展,目前正在研究多种方法来推进蛋白质测序。其中一种很有前景的方法是使用 N 端氨基酸结合剂(NAABs),它也被称为识别器,可以选择性地识别肽 N 端的氨基酸。然而,目前只有少数工程化的 NAAB 能与特定氨基酸结合,并符合生物技术试剂的要求。因此,需要鉴定和设计更多的 NAABs,以便进行可靠的鉴定,并最终进行全新的蛋白质测序。为了填补这一空白,我们对人类蛋白质 GID4 进行了改造,以创建 N 端脯氨酸(Nt-Pro)的 NAAB。虽然原生 GID4 能与 Nt-Pro 结合,但其结合力很弱(µmol/L),而且在很大程度上受 Nt-Pro 后面残基特性的影响。通过定向进化、酵母表面展示和荧光激活细胞分选,我们发现了对 Nt-Pro 结合反应更强的 GID4 序列变体。此外,带有 A252V 突变的变体在与 Nt-Pro 结合时,目标肽第二和第三位残基的影响减弱。本文概述的工作流程被证明是一种可行的 NAABs 工程设计策略,即使是从原生 Nt 结合蛋白开始,其结合也会受到 Nt 氨基酸后残基身份的强烈影响。
Engineering GID4 for use as an N-terminal proline binder via directed evolution
Nucleic acid sequencing technologies have gone through extraordinary advancements in the past several decades, significantly increasing throughput while reducing cost. To create similar advancement in proteomics, numerous approaches are being investigated to advance protein sequencing. One of the promising approaches uses N-terminal amino acid binders (NAABs), also referred to as recognizers, that selectively can identify amino acids at the N-terminus of a peptide. However, there are only a few engineered NAABs currently available that bind to specific amino acids and meet the requirements of a biotechnology reagent. Therefore, additional NAABs need to be identified and engineered to enable confident identification and, ultimately, de novo protein sequencing. To fill this gap, a human protein GID4 was engineered to create a NAAB for N-terminal proline (Nt-Pro). While native GID4 binds Nt-Pro, its binding is weak (µmol/L) and greatly influenced by the identity of residues following the Nt-Pro. Through directed evolution, yeast-surface display, and fluorescence-activated cell sorting, we identified sequence variants of GID4 with increased binding response to Nt-Pro. Moreover, variants with an A252V mutation showed a reduced influence from residues in the second and third positions of the target peptide when binding to Nt-Pro. The workflow outlined here is shown to be a viable strategy for engineering NAABs, even when starting from native Nt-binding proteins whose binding is strongly impacted by the identity of residues following Nt-amino acid.
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