Protein expression and purification最新文献_第5页

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

Monitoring of the disulfide scrambled species by mixed-mode SEC-HPLC during the purification of a bispecific antibody 在纯化双特异性抗体的过程中，通过混合模式 SEC-HPLC 监测二硫杂乱物种。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-06 DOI: 10.1016/j.pep.2024.106544

Mengying Tian , Dandan Li , Lixia Hu , Wanyuan Dong , Tongdan Wang , Yifeng Li

{"title":"Monitoring of the disulfide scrambled species by mixed-mode SEC-HPLC during the purification of a bispecific antibody","authors":"Mengying Tian , Dandan Li , Lixia Hu , Wanyuan Dong , Tongdan Wang , Yifeng Li","doi":"10.1016/j.pep.2024.106544","DOIUrl":"10.1016/j.pep.2024.106544","url":null,"abstract":"<div>Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"223 ","pages":"Article 106544"},"PeriodicalIF":1.4,"publicationDate":"2024-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555392","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

Production of stable and pure ZC3H11A – An extensively disordered RNA binding protein 生产稳定纯净的 ZC3H11A - 一种广泛紊乱的 RNA 结合蛋白。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-04 DOI: 10.1016/j.pep.2024.106542

Mostafa Fekry , Gun Stenberg , Doreen Dobritzsch , U. Helena Danielson

{"title":"Production of stable and pure ZC3H11A – An extensively disordered RNA binding protein","authors":"Mostafa Fekry , Gun Stenberg , Doreen Dobritzsch , U. Helena Danielson","doi":"10.1016/j.pep.2024.106542","DOIUrl":"10.1016/j.pep.2024.106542","url":null,"abstract":"<div>Human ZC3H11A is an RNA-binding zinc finger protein involved in mRNA export and required for the efficient growth of human nuclear replicating viruses. Its biochemical properties are largely unknown so our goal has been to produce the protein in a pure and stable form suitable for its characterization. This has been challenging since the protein is large (810 amino acids) and with only the N-terminal zinc finger domain (amino acids 1–86) being well structured, the remainder is intrinsically disordered. Our production strategies have encompassed recombinant expression of full-length, truncated and mutated ZC3H11A variants with varying purification tags and fusion proteins in several expression systems, with or without co-expression of chaperones and putative interaction partners. A range of purification schemes have been explored. Initially, only truncated ZC3H11A encompassing the zinc finger domain could successfully be produced in a stable form. It required recombinant expression in insect cells since expression in E. coli gave a protein that aggregated. To reduce problematic nucleic acid contaminations, Cys8, located in one of the zinc fingers, was substituted by Ala and Ser. Interestingly, this did not affect nucleic acid binding, but the full-length protein was stabilised while the truncated version was insoluble. Ultimately, we discovered that when using alkaline buffers (pH 9) for purification, full-length ZC3H11A expressed in Sf9 insect cells was obtained in a stable and >90 % pure form, and as a mixture of monomers, dimers, tetramers and hexamers. Many of the challenges experienced are consistent with its predicted structure and unusual charge distribution.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106542"},"PeriodicalIF":1.4,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046592824001141/pdfft?md5=9c2dece79bb1fab7f9dcbbfdc717d0f4&pid=1-s2.0-S1046592824001141-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141538535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Expression of dengue capsid-like particles in silkworm and display of envelope domain III of dengue virus serotype 2 在家蚕体内表达登革热包膜样颗粒并显示登革热病毒血清 2 型的包膜结构域 III。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-04 DOI: 10.1016/j.pep.2024.106543

Krishna Raja Muthuraman , Doddy Irawan Setyo Utomo , Mami Matsuda , Ryosuke Suzuki , Enoch Y. Park

{"title":"Expression of dengue capsid-like particles in silkworm and display of envelope domain III of dengue virus serotype 2","authors":"Krishna Raja Muthuraman , Doddy Irawan Setyo Utomo , Mami Matsuda , Ryosuke Suzuki , Enoch Y. Park","doi":"10.1016/j.pep.2024.106543","DOIUrl":"10.1016/j.pep.2024.106543","url":null,"abstract":"<div>Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106543"},"PeriodicalIF":1.4,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545170","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