Engineering cell-free systems by chemoproteomic-assisted phenotypic screening†

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY RSC Chemical Biology Pub Date : 2024-03-06 DOI:10.1039/D4CB00004H
Zarina Levitskaya, Zheng Ser, Hiromi Koh, Wang Shi Mei, Sharon Chee, Radoslaw Mikolaj Sobota and John F. Ghadessy
{"title":"Engineering cell-free systems by chemoproteomic-assisted phenotypic screening†","authors":"Zarina Levitskaya, Zheng Ser, Hiromi Koh, Wang Shi Mei, Sharon Chee, Radoslaw Mikolaj Sobota and John F. Ghadessy","doi":"10.1039/D4CB00004H","DOIUrl":null,"url":null,"abstract":"<p >Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development of cell-free protein synthesis (CFPS) technology. Phenotypic screening identified numerous compounds that enhanced protein production in yeast lysate CFPS reactions. Notably, many of these were competitive ATP kinase inhibitors, with the exploitation of their inherent substrate promiscuity redirecting ATP flux towards heterologous protein expression. Chemoproteomic-guided strain engineering partially phenocopied drug effects, with a 30% increase in protein yield observed upon deletion of the ATP-consuming SSA1 component of the HSP70 chaperone. Moreover, drug-mediated metabolic rewiring coupled with template optimization generated the highest protein yields in yeast CFPS to date using a hitherto less efficient, but more cost-effective glucose energy regeneration system. Our approach highlights the utility of target-agnostic phenotypic screening and target identification to deconvolute cell-lysate complexity, adding to the expanding repertoire of strategies for improving CFPS.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 4","pages":" 372-385"},"PeriodicalIF":4.2000,"publicationDate":"2024-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00004h?page=search","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"RSC Chemical Biology","FirstCategoryId":"1085","ListUrlMain":"https://pubs.rsc.org/en/content/articlelanding/2024/cb/d4cb00004h","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development of cell-free protein synthesis (CFPS) technology. Phenotypic screening identified numerous compounds that enhanced protein production in yeast lysate CFPS reactions. Notably, many of these were competitive ATP kinase inhibitors, with the exploitation of their inherent substrate promiscuity redirecting ATP flux towards heterologous protein expression. Chemoproteomic-guided strain engineering partially phenocopied drug effects, with a 30% increase in protein yield observed upon deletion of the ATP-consuming SSA1 component of the HSP70 chaperone. Moreover, drug-mediated metabolic rewiring coupled with template optimization generated the highest protein yields in yeast CFPS to date using a hitherto less efficient, but more cost-effective glucose energy regeneration system. Our approach highlights the utility of target-agnostic phenotypic screening and target identification to deconvolute cell-lysate complexity, adding to the expanding repertoire of strategies for improving CFPS.

Abstract Image

Abstract Image

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
通过化学蛋白组辅助表型筛选技术构建无细胞系统
表型筛选是了解和设计复杂生物系统的重要工具。我们在无细胞蛋白质合成(CFPS)技术的开发中展示了这种方法的功能。表型筛选发现了许多能在酵母裂解物 CFPS 反应中提高蛋白质产量的化合物。值得注意的是,其中许多化合物都是竞争性 ATP 激酶抑制剂,利用其固有的底物杂合性将 ATP 通量重新导向异源蛋白表达。以化学蛋白组学为指导的菌株工程学部分表征了药物效应,在删除 HSP70 合子中消耗 ATP 的 SSA1 成分后,蛋白质产量增加了 30%。此外,药物介导的新陈代谢改组与模板优化相结合,利用迄今为止效率较低但成本效益更高的葡萄糖能量再生系统,在酵母 CFPS 中产生了迄今为止最高的蛋白质产量。我们的方法凸显了靶标识别表型筛选和靶标鉴定在消除细胞裂解物复杂性方面的效用,为不断扩大的 CFPS 改进策略范围增添了新的内容。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
期刊最新文献
Back cover Cultivating the future leaders of chemical biology. Rational engineering of an antimalarial peptide with enhanced proteolytic stability and preserved parasite invasion inhibitory activity. A nanoengineered tandem nitroreductase: designing a robust prodrug-activating nanoreactor. A platform of ADAPTive scaffolds: development of CDR-H3 β-hairpin mimics into covalent inhibitors of the PD1/PDL1 immune checkpoint†
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1