DNA Ligase From Thermococcus radiotolerans: Recombinant Production, Characterization, Up-Scale Fermentation, and In Silico Evaluation.

IF 3.2 4区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Ahmed Osman, Saima Iftikhar, Bibi Nazia Murtaza, Salman Hosawi, Hisham N Tayeb, Thamir M Eid, Hayam A Alwabsi, Muhammad Shahid Nadeem
{"title":"DNA Ligase From Thermococcus radiotolerans: Recombinant Production, Characterization, Up-Scale Fermentation, and In Silico Evaluation.","authors":"Ahmed Osman, Saima Iftikhar, Bibi Nazia Murtaza, Salman Hosawi, Hisham N Tayeb, Thamir M Eid, Hayam A Alwabsi, Muhammad Shahid Nadeem","doi":"10.1002/bab.2747","DOIUrl":null,"url":null,"abstract":"<p><p>The present study describes molecular cloning, Escherichia coli expression, purification, and characterization of a DNA ligase from Thermococcus radiotolerans (TR). Optimal pH, temperature, media composition, and inducer concentrations for the production of wet cellular mass (WCM) with active enzyme have been validated. Recombinant DNA ligase-TR displayed a 44 kDa protein band on SDS-PAGE, and enzyme activity was measured at 50°C in the presence of 50 mM Tris-Cl buffer adjusted at pH 7 and supplemented with 0.4 mM ATP ± 0.1 mM NAD<sup>+</sup>. The enzyme was able to ligate two restriction products of EcoRI, independent of the presence of NAD<sup>+</sup>. The subject enzyme exhibited better activity than T4 DNA ligase. Maximum WCM was obtained at pH 7.3 and 30°C in modified M9NG medium when induced with 0.4 mM isopropyl β-d-1-thiogalactoside (IPTG). Protein sequence alignment studies have shown that DNA ligase-TR exhibits a novel and specific primary structure that is different from DNA ligases isolated from eukaryotic and other thermophilic species reported in the literature. According to in silico studies, Lys<sup>238</sup> interacts with adenosine monophosphate (AMP) and the residues responsible to engage nicked DNA include Arg<sup>41</sup>, <math> <semantics><mrow><mi>Ar</mi> <msup><mi>g</mi> <mn>43</mn></msup> </mrow> <annotation>${\\mathrm{Ar}}{{\\mathrm{g}}^{43}}$</annotation></semantics> </math> , <math> <semantics><mrow><mi>Ar</mi> <msup><mi>g</mi> <mn>50</mn></msup> </mrow> <annotation>${\\mathrm{Ar}}{{\\mathrm{g}}^{50}}$</annotation></semantics> </math> , <math> <semantics><mrow><mi>Ar</mi> <msup><mi>g</mi> <mn>51</mn></msup> </mrow> <annotation>${\\mathrm{Ar}}{{\\mathrm{g}}^{51}}$</annotation></semantics> </math> , <math> <semantics><mrow><mi>Ar</mi> <msup><mi>g</mi> <mn>267</mn></msup> </mrow> <annotation>${\\mathrm{Ar}}{{\\mathrm{g}}^{267}}$</annotation></semantics> </math> , <math> <semantics><mrow><mi>Ar</mi> <msup><mi>g</mi> <mn>270</mn></msup> </mrow> <annotation>${\\mathrm{Ar}}{{\\mathrm{g}}^{270}}$</annotation></semantics> </math> , and Arg<sup>367</sup>. About 51% secondary structure consists of α-helices and β-pleated sheets. High-temperature stability, better half-life, and high activity of DNA ligase-TR advocate its suitability in the DNA manipulation procedures.</p>","PeriodicalId":9274,"journal":{"name":"Biotechnology and applied biochemistry","volume":" ","pages":"e2747"},"PeriodicalIF":3.2000,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology and applied biochemistry","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1002/bab.2747","RegionNum":4,"RegionCategory":"生物学","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

The present study describes molecular cloning, Escherichia coli expression, purification, and characterization of a DNA ligase from Thermococcus radiotolerans (TR). Optimal pH, temperature, media composition, and inducer concentrations for the production of wet cellular mass (WCM) with active enzyme have been validated. Recombinant DNA ligase-TR displayed a 44 kDa protein band on SDS-PAGE, and enzyme activity was measured at 50°C in the presence of 50 mM Tris-Cl buffer adjusted at pH 7 and supplemented with 0.4 mM ATP ± 0.1 mM NAD+. The enzyme was able to ligate two restriction products of EcoRI, independent of the presence of NAD+. The subject enzyme exhibited better activity than T4 DNA ligase. Maximum WCM was obtained at pH 7.3 and 30°C in modified M9NG medium when induced with 0.4 mM isopropyl β-d-1-thiogalactoside (IPTG). Protein sequence alignment studies have shown that DNA ligase-TR exhibits a novel and specific primary structure that is different from DNA ligases isolated from eukaryotic and other thermophilic species reported in the literature. According to in silico studies, Lys238 interacts with adenosine monophosphate (AMP) and the residues responsible to engage nicked DNA include Arg41, Ar g 43 ${\mathrm{Ar}}{{\mathrm{g}}^{43}}$ , Ar g 50 ${\mathrm{Ar}}{{\mathrm{g}}^{50}}$ , Ar g 51 ${\mathrm{Ar}}{{\mathrm{g}}^{51}}$ , Ar g 267 ${\mathrm{Ar}}{{\mathrm{g}}^{267}}$ , Ar g 270 ${\mathrm{Ar}}{{\mathrm{g}}^{270}}$ , and Arg367. About 51% secondary structure consists of α-helices and β-pleated sheets. High-temperature stability, better half-life, and high activity of DNA ligase-TR advocate its suitability in the DNA manipulation procedures.

求助全文
约1分钟内获得全文 求助全文
来源期刊
Biotechnology and applied biochemistry
Biotechnology and applied biochemistry 工程技术-生化与分子生物学
CiteScore
6.00
自引率
7.10%
发文量
117
审稿时长
3 months
期刊介绍: Published since 1979, Biotechnology and Applied Biochemistry is dedicated to the rapid publication of high quality, significant research at the interface between life sciences and their technological exploitation. The Editors will consider papers for publication based on their novelty and impact as well as their contribution to the advancement of medical biotechnology and industrial biotechnology, covering cutting-edge research in synthetic biology, systems biology, metabolic engineering, bioengineering, biomaterials, biosensing, and nano-biotechnology.
×
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
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
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 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信