底物结合中温度依赖的亲和力变化影响BthC2c1的裂解活性。

IF 1 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Protein and Peptide Letters Pub Date : 2023-01-01 DOI:10.2174/0929866530666230125100320
Dan Wu, Jieting Liu, Yong Liu, Yufei Qiu, Zhiqin Cao, Yu Pan, Jiayi Shi, Xiaohuan Yuan
{"title":"底物结合中温度依赖的亲和力变化影响BthC2c1的裂解活性。","authors":"Dan Wu,&nbsp;Jieting Liu,&nbsp;Yong Liu,&nbsp;Yufei Qiu,&nbsp;Zhiqin Cao,&nbsp;Yu Pan,&nbsp;Jiayi Shi,&nbsp;Xiaohuan Yuan","doi":"10.2174/0929866530666230125100320","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>The CRISPR-Cas system is an adaptive immune mechanism for bacteria and archaea to resist foreign invasion. Currently, Cas9 and Cpf1 have been widely studied and applied in gene editing. C2c1 is a newly discovered CRISPR-Cas system endonuclease. It has broad application prospects due to its small molecular weight and high substrate recognition specificity.</p><p><strong>Objectives: </strong>Bacillus thermoamylovorans C2c1(BthC2c1) was expressed in E. coli C43 (DE3) competent cells, purified, and the BthC2c1-sgRNA-dsDNA complex was assembled. The effect of temperature on the cleavage ability of the BthC2c1 system was investigated.</p><p><strong>Methods: </strong>The cDNA of BthC2c1 was cloned into the vector pGEX-6P-1. BthC2c1 was expressed in E. coli C43(DE3) cells and purified using a GST affinity column and FPLC. The sgRNAs were transcribed and purified in vitro, and the complexes were assembled by gel filtration chromatography. The enzyme cleavage activity of BthC2c1 at different temperatures was investigated using an in vitro cleavage assay. Microscale Thermophoresis detected the affinity of the BthC2c1-sgRNA complexes to substrate DNA.</p><p><strong>Results: </strong>BthC2c1 proteins were prokaryotically expressed and purified. The complex of BthC2c1 with sgRNA and dsDNA was assembled. In vitro cleavage assay results showed that BthC2c1 cleaved the target DNA at temperatures ranging from 37°C to 67°C. The cleavage ability of BthC2c1 at 42<sup>o</sup>C was stronger than that at 37<sup>o</sup>C. The results of affinity detection showed that the affinity between the BthC2c1-sgRNA complex and ds36/36 at 42<sup>o</sup>C was stronger than that at 37<sup>o</sup>C.</p><p><strong>Conclusion: </strong>In this study, BthC2c1 was expressed, purified, and assembled into a complex with sgRNA and dsDNA. BthC2c1 cleaved DNA within the temperature range of 37<sup>o</sup>C to 67<sup>o</sup>C. The affinity of BthC2c1-sgRNA to DNA at 42°C was significantly enhanced than that at 37°C. It may be related to its stringent substrate recognition pattern, which differs from Cas9 and Cpf1. The temperature-dependent affinity changes of substrate binding may be part of the reason for the stronger cleavage activity of BthC2c1 at 42<sup>o</sup>C. This study may provide an experimental basis for optimizing and modifying the C2c1 gene editing system.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":"30 3","pages":"233-241"},"PeriodicalIF":1.0000,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10249141/pdf/","citationCount":"0","resultStr":"{\"title\":\"Temperature-Dependent Affinity Changes in Substrate Binding Affect the Cleavage Activity of BthC2c1.\",\"authors\":\"Dan Wu,&nbsp;Jieting Liu,&nbsp;Yong Liu,&nbsp;Yufei Qiu,&nbsp;Zhiqin Cao,&nbsp;Yu Pan,&nbsp;Jiayi Shi,&nbsp;Xiaohuan Yuan\",\"doi\":\"10.2174/0929866530666230125100320\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>The CRISPR-Cas system is an adaptive immune mechanism for bacteria and archaea to resist foreign invasion. Currently, Cas9 and Cpf1 have been widely studied and applied in gene editing. C2c1 is a newly discovered CRISPR-Cas system endonuclease. It has broad application prospects due to its small molecular weight and high substrate recognition specificity.</p><p><strong>Objectives: </strong>Bacillus thermoamylovorans C2c1(BthC2c1) was expressed in E. coli C43 (DE3) competent cells, purified, and the BthC2c1-sgRNA-dsDNA complex was assembled. The effect of temperature on the cleavage ability of the BthC2c1 system was investigated.</p><p><strong>Methods: </strong>The cDNA of BthC2c1 was cloned into the vector pGEX-6P-1. BthC2c1 was expressed in E. coli C43(DE3) cells and purified using a GST affinity column and FPLC. The sgRNAs were transcribed and purified in vitro, and the complexes were assembled by gel filtration chromatography. The enzyme cleavage activity of BthC2c1 at different temperatures was investigated using an in vitro cleavage assay. Microscale Thermophoresis detected the affinity of the BthC2c1-sgRNA complexes to substrate DNA.</p><p><strong>Results: </strong>BthC2c1 proteins were prokaryotically expressed and purified. The complex of BthC2c1 with sgRNA and dsDNA was assembled. In vitro cleavage assay results showed that BthC2c1 cleaved the target DNA at temperatures ranging from 37°C to 67°C. The cleavage ability of BthC2c1 at 42<sup>o</sup>C was stronger than that at 37<sup>o</sup>C. The results of affinity detection showed that the affinity between the BthC2c1-sgRNA complex and ds36/36 at 42<sup>o</sup>C was stronger than that at 37<sup>o</sup>C.</p><p><strong>Conclusion: </strong>In this study, BthC2c1 was expressed, purified, and assembled into a complex with sgRNA and dsDNA. BthC2c1 cleaved DNA within the temperature range of 37<sup>o</sup>C to 67<sup>o</sup>C. The affinity of BthC2c1-sgRNA to DNA at 42°C was significantly enhanced than that at 37°C. It may be related to its stringent substrate recognition pattern, which differs from Cas9 and Cpf1. The temperature-dependent affinity changes of substrate binding may be part of the reason for the stronger cleavage activity of BthC2c1 at 42<sup>o</sup>C. This study may provide an experimental basis for optimizing and modifying the C2c1 gene editing system.</p>\",\"PeriodicalId\":20736,\"journal\":{\"name\":\"Protein and Peptide Letters\",\"volume\":\"30 3\",\"pages\":\"233-241\"},\"PeriodicalIF\":1.0000,\"publicationDate\":\"2023-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10249141/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Protein and Peptide Letters\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.2174/0929866530666230125100320\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein and Peptide Letters","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.2174/0929866530666230125100320","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0

摘要

背景:CRISPR-Cas系统是细菌和古细菌抵抗外来入侵的适应性免疫机制。目前,Cas9和Cpf1在基因编辑中得到了广泛的研究和应用。C2c1是一种新发现的CRISPR-Cas系统内切酶。其分子量小、底物识别特异性高,具有广阔的应用前景。目的:在大肠杆菌C43 (DE3)感受态细胞中表达热淀粉样芽孢杆菌C2c1(BthC2c1),纯化并组装BthC2c1- sgrna - dsdna复合物。研究了温度对BthC2c1体系解理能力的影响。方法:将BthC2c1 cDNA克隆到载体pGEX-6P-1中。BthC2c1在大肠杆菌C43(DE3)细胞中表达,并通过GST亲和柱和FPLC纯化。对sgrna进行体外转录和纯化,并用凝胶过滤层析法组装复合物。采用体外裂解实验研究了BthC2c1在不同温度下的酶裂解活性。微尺度热泳检测BthC2c1-sgRNA复合物与底物DNA的亲和力。结果:BthC2c1蛋白得到原核表达和纯化。组装BthC2c1与sgRNA和dsDNA的复合物。体外裂解实验结果表明,BthC2c1在37 ~ 67℃的温度范围内可裂解靶DNA。BthC2c1在42℃时的裂解能力强于37℃时。亲和力检测结果显示,BthC2c1-sgRNA复合物与ds36/36在42℃时的亲和力比在37℃时强。结论:在本研究中,BthC2c1被表达、纯化并与sgRNA和dsDNA组装成复合物。BthC2c1在37℃至67℃的温度范围内切割DNA。BthC2c1-sgRNA在42°C时对DNA的亲和力比在37°C时显著增强。这可能与它严格的底物识别模式不同于Cas9和Cpf1有关。BthC2c1在42℃时具有较强的裂解活性,其与底物结合的亲和力随温度的变化可能是原因之一。本研究可为C2c1基因编辑系统的优化和修饰提供实验依据。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

摘要图片

摘要图片

摘要图片

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
Temperature-Dependent Affinity Changes in Substrate Binding Affect the Cleavage Activity of BthC2c1.

Background: The CRISPR-Cas system is an adaptive immune mechanism for bacteria and archaea to resist foreign invasion. Currently, Cas9 and Cpf1 have been widely studied and applied in gene editing. C2c1 is a newly discovered CRISPR-Cas system endonuclease. It has broad application prospects due to its small molecular weight and high substrate recognition specificity.

Objectives: Bacillus thermoamylovorans C2c1(BthC2c1) was expressed in E. coli C43 (DE3) competent cells, purified, and the BthC2c1-sgRNA-dsDNA complex was assembled. The effect of temperature on the cleavage ability of the BthC2c1 system was investigated.

Methods: The cDNA of BthC2c1 was cloned into the vector pGEX-6P-1. BthC2c1 was expressed in E. coli C43(DE3) cells and purified using a GST affinity column and FPLC. The sgRNAs were transcribed and purified in vitro, and the complexes were assembled by gel filtration chromatography. The enzyme cleavage activity of BthC2c1 at different temperatures was investigated using an in vitro cleavage assay. Microscale Thermophoresis detected the affinity of the BthC2c1-sgRNA complexes to substrate DNA.

Results: BthC2c1 proteins were prokaryotically expressed and purified. The complex of BthC2c1 with sgRNA and dsDNA was assembled. In vitro cleavage assay results showed that BthC2c1 cleaved the target DNA at temperatures ranging from 37°C to 67°C. The cleavage ability of BthC2c1 at 42oC was stronger than that at 37oC. The results of affinity detection showed that the affinity between the BthC2c1-sgRNA complex and ds36/36 at 42oC was stronger than that at 37oC.

Conclusion: In this study, BthC2c1 was expressed, purified, and assembled into a complex with sgRNA and dsDNA. BthC2c1 cleaved DNA within the temperature range of 37oC to 67oC. The affinity of BthC2c1-sgRNA to DNA at 42°C was significantly enhanced than that at 37°C. It may be related to its stringent substrate recognition pattern, which differs from Cas9 and Cpf1. The temperature-dependent affinity changes of substrate binding may be part of the reason for the stronger cleavage activity of BthC2c1 at 42oC. This study may provide an experimental basis for optimizing and modifying the C2c1 gene editing system.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
Protein and Peptide Letters
Protein and Peptide Letters 生物-生化与分子生物学
CiteScore
2.90
自引率
0.00%
发文量
98
审稿时长
2 months
期刊介绍: Protein & Peptide Letters publishes letters, original research papers, mini-reviews and guest edited issues in all important aspects of protein and peptide research, including structural studies, advances in recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, and drug design. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallization and preliminary structure determination of biologically important proteins are considered only if they include significant new approaches or deal with proteins of immediate importance, and preliminary structure determinations of biologically important proteins. Purely theoretical/review papers should provide new insight into the principles of protein/peptide structure and function. Manuscripts describing computational work should include some experimental data to provide confirmation of the results of calculations. Protein & Peptide Letters focuses on: Structure Studies Advances in Recombinant Expression Drug Design Chemical Synthesis Function Pharmacology Enzymology Conformational Analysis Immunology Biotechnology Protein Engineering Protein Folding Sequencing Molecular Recognition Purification and Analysis
期刊最新文献
Recombinant Production of Ib-AMP4 and Oncorhyncin II Antimicrobial Peptides and Antimicrobial Synergistic Assessment on the Treatment of Staphylococcus aureus Under in vitro Condition. Overexpression of HIF2α Enhances the Angiogenesis-Promoting Effect of hUC-MSC-Derived Extracellular Vesicles by Stimulating miR-146a. Recent Trends in Development of Novel Therapeutics for Modulation of 14-3-3 Protein-Protein Interactions in Diseases. Leptin/Melanocortin Pathway in Cholelithiasis Patients: A Diagnostic Perspective? Exploring the Therapeutic Potential of Noncoding RNAs in Alzheimer's Disease.
×
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