Protein expression and purification最新文献_第10页

A fast and simple automated multi-step protein purification method for ÄKTA go systems 用于 ÄKTA go 系统的快速、简单的自动化多步蛋白质纯化方法。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-31 DOI: 10.1016/j.pep.2024.106560

Daichi Morimoto , Erik Walinda

{"title":"A fast and simple automated multi-step protein purification method for ÄKTA go systems","authors":"Daichi Morimoto , Erik Walinda","doi":"10.1016/j.pep.2024.106560","DOIUrl":"10.1016/j.pep.2024.106560","url":null,"abstract":"<div>Automation of protein purification methods can increase researchers' efficiency in life sciences. However, currently reported automated protein purification methods require cost-intensive fast protein liquid chromatography systems, such as ÄKTA pure and ÄKTA explorer, without any reported application to the more cost-efficient entry-level system, ÄKTA go. To fill this gap, here we propose a fast, efficient, and versatile automated protein purification strategy for the ÄKTA go. Straightforward integration of two additional accessories, a column valve and a sample loop, into the default ÄKTA go system and making minor rearrangements of flow lines, enabled automation of multi-step protein purification processes. Utilizing this established system, we demonstrate the automated purification of three distinct types of proteins: ubiquitin, polyhistidine-tagged talin, and GST-tagged human rhinovirus 14 3C protease. The described automation strategy is suitable even for small budget-conscious laboratories operating on ÄKTA go systems, thus reducing researchers’ time and efforts spent on routine sample preparation tasks of their investigations.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"223 ","pages":"Article 106560"},"PeriodicalIF":1.4,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141879317","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Improving the catalytic activity and thermostability of Aspergillus niger xylanase through computational design 通过计算设计提高黑曲霉木聚糖酶的催化活性和热稳定性。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-31 DOI: 10.1016/j.pep.2024.106561

Shuyan Duan , Yaoyao Wu , Tianzhu Chao , Nan Zhang , Zhaoyi Wei , Rui Ji

{"title":"Improving the catalytic activity and thermostability of Aspergillus niger xylanase through computational design","authors":"Shuyan Duan , Yaoyao Wu , Tianzhu Chao , Nan Zhang , Zhaoyi Wei , Rui Ji","doi":"10.1016/j.pep.2024.106561","DOIUrl":"10.1016/j.pep.2024.106561","url":null,"abstract":"<div>Xylanase plays the most important role in catalyzing xylan to xylose moieties. GH11 xylanases have been widely used in many fields, but most GH11 xylanases are mesophilic enzymes. To improve the catalytic activity and thermostability of Aspergillus niger xylanase (Xyn-WT), we predicted potential key mutation sites of Xyn-WT through multiple computer-aided enzyme engineering strategies. We introduce a simple and economical Ni affinity chromatography purification method to obtain high-purity xylanase and its mutants. Ten mutants (Xyn-A, Xyn-B, Xyn-C, E45T, Q93R, E45T/Q93R, A161P, Xyn-D, Xyn-E, Xyn-F) were identified. Among the ten mutants, four (Xyn-A, Xyn-C, A161P, Xyn-F) presented improved thermal stability and activity, with Xyn-F(A161P/E45T/Q93R) being the most thermally stable and active. Compared with Xyn-WT, after heat treatment at 55 °C and 60 °C for 10 min, the remaining enzyme activity of Xyn-F was 12 and 6 times greater than that of Xyn-WT, respectively, and Xyn-F was approximately 1.5 times greater than Xyn-WT when not heat treated. The pH adaptation of Xyn-F was also significantly enhanced. In summary, an improved catalytic activity and thermostability of the design variant Xyn-F has been reported.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"223 ","pages":"Article 106561"},"PeriodicalIF":1.4,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141879319","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Biochemical characterization of a high affinity phosphate transporter (PiPT) from root endophyte fungus Piriformospora indica 根内生真菌 Piriformospora indica 的高亲和性磷酸盐转运体（PiPT）的生化特征。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-31 DOI: 10.1016/j.pep.2024.106559

Hemant Kumar , Aayushi Bajaj , Paras Kumar , Rupesh Aggarwal , Vinayak Chalia , Rajesh Kumar Pradhan , Ritu Yadav , Shalini Sinha , Vishad Agarwal , William Harries , Meenakshi Dua , Robert M. Stroud , Atul Kumar Johri

引用次数: 0

Identification of chitinase from Bacillus velezensis strain S161 and its antifungal activity against Penicillium digitatum 鉴定 Velezensis 杆菌 S161 菌株的几丁质酶及其对数字青霉的抗真菌活性。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-31 DOI: 10.1016/j.pep.2024.106562

Feng Liu , Song Chen , Xingbang Chen , Bin Yong , Bing He

{"title":"Identification of chitinase from Bacillus velezensis strain S161 and its antifungal activity against Penicillium digitatum","authors":"Feng Liu , Song Chen , Xingbang Chen , Bin Yong , Bing He","doi":"10.1016/j.pep.2024.106562","DOIUrl":"10.1016/j.pep.2024.106562","url":null,"abstract":"<div>Previous studies have demonstrated the presence of chitinase in Bacillus velezensis through extensive genomic sequencing and experimental analyses. However, the detailed structure, functional roles, and antifungal activity of these chitinases remain poorly characterized. In this study, genomic screening identified three genes—chiA, chiB, and lpmo10—associated with chitinase degradation in B. velezensis S161. These genes encode chitinases ChiA and ChiB, and lytic polysaccharide monooxygenase LPMO10. Both ChiA and ChiB contain two CBM50 binding domains and one catalytic domain, whereas LPMO10 includes a signal peptide and a single catalytic domain. The chitinases ChiA, its truncated variant ChiA2, and ChiB were heterologously expressed in Escherichia coli. The purified enzymes efficiently degraded colloidal chitin and inhibited the spore germination of Penicillium digitatum. Notably, even after losing one CBM50 domain, the resultant enzyme, consisting of the remaining CBM50 domain and the catalytic domain, maintained its colloidal chitin hydrolysis and antifungal activity, indicating commendable stability. These results underscore the role of B. velezensis chitinases in suppressing plant pathogenic fungi and provide a solid foundation for developing and applying chitinase-based biocontrol strategies.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"223 ","pages":"Article 106562"},"PeriodicalIF":1.4,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141879318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Efficient heterologous expression of cellobiose 2-epimerase gene in Escherichia coli under the control of T7 lac promoter without addition of IPTG and lactose 在 T7 lac 启动子控制下，无需添加 IPTG 和乳糖，在大肠杆菌中高效异源表达纤维生物糖 2-epimerase 基因。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-27 DOI: 10.1016/j.pep.2024.106558

Shuzhen Li , Wei Shen , Yuanyuan Xia , Xianzhong Chen , Haiquan Yang

{"title":"Efficient heterologous expression of cellobiose 2-epimerase gene in Escherichia coli under the control of T7 lac promoter without addition of IPTG and lactose","authors":"Shuzhen Li , Wei Shen , Yuanyuan Xia , Xianzhong Chen , Haiquan Yang","doi":"10.1016/j.pep.2024.106558","DOIUrl":"10.1016/j.pep.2024.106558","url":null,"abstract":"<div>In this study, the cellobiose 2-epimerase gene csce from Caldicellulosiruptor saccharolyticus was expressed in Escherichia coli using TB medium containing yeast extract Oxoid and tryptone Oxoid. Interesting, it was found that when the concentration of isopropyl-beta-d-thiogalactopyranoside (IPTG) and lactose was 0 (no addition), the activity of cellobiose 2-epimerase reached 5.88 U/mL. It was 3.70-fold higher than the activity observed when 1.0 mM IPTG was added. When using M9 medium without yeast extract Oxoid and tryptone Oxoid, cellobiose 2-epimerase gene could not be expressed without IPTG and lactose. However, cellobiose 2-epimerase gene could be expressed when yeast extract Oxoid or tryptone Oxoid was added, indicating that these supplements contained inducers for gene expression. In the absence of IPTG and lactose, the addition of soy peptone Angel-1 or yeast extract Angel-1 to M9 medium significantly upregulated the expression of cellobiose 2-epimerase gene in E. coli BL21 pET28a-csce, and these inductions led to higher expression levels compared to tryptone Oxoid or yeast extract Oxoid. The relative transcription level of csce was consistent with its expression level in E. coli BL21 pET28a-csce. In the medium TB without IPTG and lactose and containing yeast extract Angel-1 and soy peptone Angel-1, the activity of cellobiose 2-epimerase reached 6.88 U/mL, representing a 2.2-fold increase compared to previously reported maximum activity in E. coli. The significance of this study lies in its implications for efficient heterologous expression of recombinant enzyme proteins in E. coli without the need for IPTG and lactose addition.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"223 ","pages":"Article 106558"},"PeriodicalIF":1.4,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141793189","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Diverse approaches to express recombinant spike protein: A comprehensive review 表达重组尖峰蛋白的多种方法：全面回顾。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-14 DOI: 10.1016/j.pep.2024.106556

Jk Nithya Shree, T. Premika, S. Sharlin, A. Annie Aglin

引用次数: 0

The biochemical characterization of a TatD nuclease from Thermus thermophilus 嗜热菌 TatD 核酸酶的生化特征。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-14 DOI: 10.1016/j.pep.2024.106557

Yi-Xuan Zhao , Xiao Xiang , Xi-Peng Liu

{"title":"The biochemical characterization of a TatD nuclease from Thermus thermophilus","authors":"Yi-Xuan Zhao , Xiao Xiang , Xi-Peng Liu","doi":"10.1016/j.pep.2024.106557","DOIUrl":"10.1016/j.pep.2024.106557","url":null,"abstract":"<div>Nucleases play pivotal roles in DNA repair and apoptosis. Moreover, they have various applications in biotechnology and industry. Among nucleases, TatD has been characterized as an exonuclease with various biological functions in different organisms. Here, we biochemically characterized the potential TatD nuclease from Thermus thermophilus. The tatD gene from T. thermophilus was cloned, then the recombinant TatD nuclease was expressed and purified. Our results revealed that the TthTatD nuclease could degrade both single-stranded and double-stranded DNA, and its activity is dependent on the divalent metal ions Mg2+ and Mn2+. Remarkably, the activity of TthTatD nuclease is highest at 37 °C and decreases with increasing temperature. TthTatD is not a thermostable enzyme, even though it is from a thermophilic bacterium. Based on the sequence similarity and molecular docking of the DNA substrate into the modeled TthTatD structure, several key conserved residues were identified and their roles were confirmed by analyzing the enzymatic activities of the site-directed mutants. The residues E86 and H149 play key roles in binding metal ions, residues R124/K126 and K211/R212 had a critical role in binding DNA substrate. Our results confirm the enzymatic properties of TthTatD and provide a primary basis for its possible application in biotechnology.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"223 ","pages":"Article 106557"},"PeriodicalIF":1.4,"publicationDate":"2024-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141620792","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Purification of α-lactalbumin and β-lactoglobulin from cow milk 从牛奶中提纯 α-乳白蛋白和 β-乳球蛋白。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-14 DOI: 10.1016/j.pep.2024.106555

Kimia Ahadi-Amandi , Seyyed Abolghasem Ghadami , Narges Sayari , Reza Khodarahmi

引用次数: 0

Preparation of recombinant neuritin protein 制备重组神经肽蛋白。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-11 DOI: 10.1016/j.pep.2024.106554

Pingping Meng , Liyan Zhu , Jiatong Guo , Yuanyuan Li , Yu Wei , Jiawei Sun , Jingling Zhu

{"title":"Preparation of recombinant neuritin protein","authors":"Pingping Meng , Liyan Zhu , Jiatong Guo , Yuanyuan Li , Yu Wei , Jiawei Sun , Jingling Zhu","doi":"10.1016/j.pep.2024.106554","DOIUrl":"10.1016/j.pep.2024.106554","url":null,"abstract":"<div>Neuritin plays an important role in promoting nerve injury repair and maintaining synaptic plasticity, making it a potential therapeutic target for the treatment of nerve injury and neurodegenerative diseases. The present study aimed to obtain an active, unlabeled neuritin protein. Initially, a neuritin protein expression system with an enterokinase site was constructed in Escherichia coli. After optimizing induction conditions and screening for high expression, a neuritin recombinant protein with purity exceeding 85 % was obtained through Ni-affinity chromatography. Subsequently, unlabeled neuritin with a molecular weight of 11 kDa was obtained through the enzymatic cleavage of the His label using an enterokinase. Furthermore, a neuritin recombinant protein with purity exceeding 95 % was obtained using gel chromatography. Functional investigations revealed that neurite outgrowth of PC12 cells was stimulated by the isolated neuritin. This study establishes a method to obtain active and unlabeled neuritin protein, providing a foundation for subsequent research on its biological functions.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"223 ","pages":"Article 106554"},"PeriodicalIF":1.4,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141604057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Cloning and characterization of a hyaluronate lyase EsHyl8 from Escherichia sp. A99 来自 Escherichia sp. A99 的透明质酸裂解酶 EsHyl8 的克隆和表征

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-10 DOI: 10.1016/j.pep.2024.106551

Xiuli Cui , Zheng Fu , Hainan Wang , Wengong Yu , Feng Han

引用次数: 0