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
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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.

来自耐辐射热球菌的DNA连接酶:重组生产,表征,大规模发酵和硅评价。
本研究描述了分子克隆、大肠杆菌表达、纯化和鉴定放射耐药热球菌(TR) DNA连接酶。最佳的pH,温度,培养基组成和诱导剂浓度的生产湿细胞团块(WCM)与活性酶已被验证。重组DNA连接酶- tr在SDS-PAGE上显示一个44 kDa的蛋白带,酶活性在50°C下测定,存在50 mM Tris-Cl缓冲液,pH调节为7,添加0.4 mM ATP±0.1 mM NAD+。该酶能够连接EcoRI的两个限制性内切产物,独立于NAD+的存在。该酶的活性优于T4 DNA连接酶。以0.4 mM异丙基β-d-1-硫代半乳糖苷(IPTG)诱导,在pH 7.3、30℃条件下,WCM最大。蛋白质序列比对研究表明,DNA连接酶tr表现出一种新的特异性初级结构,与文献报道的真核生物和其他嗜热物种分离的DNA连接酶不同。根据硅片研究,Lys238与单磷酸腺苷(AMP)相互作用,负责接合有缺陷DNA的残基包括Arg41、arg 43 ${\ mathm {Ar}}{\ mathm {g}}^{43}}$、arg 50 ${\ mathm {Ar}}{\ mathm {g}}^{50}}$、arg 51 ${\ mathm {Ar}}{\ mathm {g}}^{51}}$、arg 267 ${\ mathm {Ar}}{\ mathm {g}}}^{267}}$、arg 270 ${\ mathm {Ar}}{{\ mathm {g}}}^{270} $和Arg367。约51%的二级结构由α-螺旋和β-褶片状组成。DNA连接酶- tr具有高温稳定性、较好的半衰期和较高的活性,适合应用于DNA操作。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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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.
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