Protein expression and purification最新文献_第4页

Optimized vector for functional expression of the human bitter taste receptor TAS2R14 in HEK293 cells 人苦味受体TAS2R14在HEK293细胞中功能表达的优化载体。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-10 DOI: 10.1016/j.pep.2024.106643

Christine Belloir, Adèle Gautier, Adeline Karolkowski, Thomas Delompré, Mathilde Jeannin, Lucie Moitrier, Fabrice Neiers, Loïc Briand

{"title":"Optimized vector for functional expression of the human bitter taste receptor TAS2R14 in HEK293 cells","authors":"Christine Belloir, Adèle Gautier, Adeline Karolkowski, Thomas Delompré, Mathilde Jeannin, Lucie Moitrier, Fabrice Neiers, Loïc Briand","doi":"10.1016/j.pep.2024.106643","DOIUrl":"10.1016/j.pep.2024.106643","url":null,"abstract":"<div><div>Bitter is one of the five basic taste qualities, along with salty, sour, sweet and umami, used by mammals to access the quality of their food and orient their eating behaviour. Bitter taste detection prevents the ingestion of food potentially contaminated by bitter-tasting toxins. Bitter taste perception is mediated by a family of G protein-coupled receptors (GPCRs) called TAS2Rs. Humans possess 25 TAS2Rs (human type II taste receptors), enabling the detection of thousands of chemically diverse bitter compounds. The identification of agonists/antagonists and molecular mechanisms that govern receptor-ligand interaction has been primarily achieved through functional expression of TAS2Rs in heterologous cells. However, TAS2R receptors, like many other GPCRs, suffer from marginal cell surface expression. In this study, we compared the functionality of 9 engineered chimeric receptors, focusing our experiments on TAS2R14, a broadly tuned receptor that recognizes over 151 identified compounds. Among the different tested signal peptides, rat somatostatin receptor subtype 3 results in higher potency of aristolochic acid-induced calcium signalling than other tested export tags, such as bovine rhodopsin, murine Igκ-chain or human mGluR5. The addition of a MAX sequence enhances both TAS2R14 potency and efficacy. We also confirm that the FLAG epitope, when located at the C-terminal, interferes less with the TAS2R14 functionality, enabling reliable evaluation of this receptor at the cell surface using immunohistochemistry. Finally, these observations are also confirmed for TAS2R14 and TAS1R2/TAS1R3 (the sweet taste receptor) stimulated by 12 bitter compounds and by sucralose and neotame, respectively.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106643"},"PeriodicalIF":1.4,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142819017","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

Optimized extraction, identification and characterization of the mosquitocidal surface layer protein from a local bacterial isolate Lysinibacillus sphaericus Q001 球形赖氨酸芽孢杆菌Q001杀蚊表层蛋白的优化提取、鉴定及特性研究。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-09 DOI: 10.1016/j.pep.2024.106639

Hamayun Arshad , Qurratulann Afza Gardner , Saira Ahmad , Syeda Sadia Bukhari , Muhammad Akhtar

{"title":"Optimized extraction, identification and characterization of the mosquitocidal surface layer protein from a local bacterial isolate Lysinibacillus sphaericus Q001","authors":"Hamayun Arshad , Qurratulann Afza Gardner , Saira Ahmad , Syeda Sadia Bukhari , Muhammad Akhtar","doi":"10.1016/j.pep.2024.106639","DOIUrl":"10.1016/j.pep.2024.106639","url":null,"abstract":"<div><div>Surface layer (S-layer) is an extracellular proteinous layer consisting of two-dimensional lattice. It is typically present on archaea and also found on some bacteria. S-layer proteins from some bacteria are reported to be toxic to mosquito larvae. Here, we aimed to extract and characterize the surface layer protein from a local bacterial strain named <em>Lysinibacillus sphaericus</em> Q001. This bacterium was isolated from Pakistan and characterized through various biochemical tests. It was identified as <em>Lysinibacillus sphaericus</em> through 16S rRNA ribotyping (NCBI accession no. OQ701385.1) and matrix-assisted laser desorption/ionization (MALDI) biotyping with 2.18 ± 0.059 score. The S-layer protein was extracted by both cation exchange method and guanidinium chloride extraction method. The optimized method for the extraction and purification of S-layer yielded 35 mg of protein from 1 L culture of <em>L. sphaericus</em> Q001. A potential S-layer protein band (120 kDa) detected by SDS-PAGE was confirmed by bottom-up proteomics i.e., in-gel tryptic digestion of the protein followed by MALDI-TOF analysis and peptide mass fingerprinting (PMF). The insecticidal bioassays revealed that S-layer protein of <em>L. sphaericus</em> Q001 was toxic against <em>Aedes aegypti</em> larvae with LC<sub>50</sub> value of 11 μg/ml. This shows its potential to be used as an alternative to chemical larvicides.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106639"},"PeriodicalIF":1.4,"publicationDate":"2024-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142786779","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 and compositional analysis of full-length influenza virus hemagglutinin in Nanodiscs: Insights from multi-angle light scattering 纳米盘中全长流感病毒血凝素的生产和成分分析：多角度光散射的启示。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-07 DOI: 10.1016/j.pep.2024.106641

Tim G.J. Knetsch, Marcellus Ubbink

{"title":"Production and compositional analysis of full-length influenza virus hemagglutinin in Nanodiscs: Insights from multi-angle light scattering","authors":"Tim G.J. Knetsch, Marcellus Ubbink","doi":"10.1016/j.pep.2024.106641","DOIUrl":"10.1016/j.pep.2024.106641","url":null,"abstract":"<div><div>The global threat of pandemics highlights the urgency of developing innovative vaccine strategies. Viral spike proteins are the primary antigens recognized by the immune system and serve as key targets for vaccine development. This study reports the production of full-length Influenza A virus surface glycoprotein, hemagglutinin (HA), and its incorporation into Nanodiscs (NDs). HA was expressed in insect cells and purified using detergents, maintaining its functional integrity. Characterisation by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) confirmed that HA could be incorporated into 1-palmitoyl-2-oleoyl-<em>sn</em>-glycero-3-phosphocholine (POPC) NDs as a single trimer. SEC-MALS was instrumental in analysing the composition of NDs, which included HA, membrane scaffold proteins, lipids, and glycans. These findings provide a robust framework for the production and reconstitution of glycoproteins in NDs, and offers valuable insights into the study of multi-component nanoparticles using MALS. Our work highlights the potential of NDs for studying viral glycoproteins and advances the development of well-defined recombinant ND-based vaccines.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106641"},"PeriodicalIF":1.4,"publicationDate":"2024-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142802104","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

The N-terminal polypeptide of a new shell matrix protein hicraqin accelerates the rate of calcium carbonate deposition 新壳基质蛋白hicraqin的n端多肽加速了碳酸钙沉积的速度。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-06 DOI: 10.1016/j.pep.2024.106642

Chenchen Liang , Jiali Liu , Guiling Wang , Xiaojun Liu

{"title":"The N-terminal polypeptide of a new shell matrix protein hicraqin accelerates the rate of calcium carbonate deposition","authors":"Chenchen Liang , Jiali Liu , Guiling Wang , Xiaojun Liu","doi":"10.1016/j.pep.2024.106642","DOIUrl":"10.1016/j.pep.2024.106642","url":null,"abstract":"<div><div>Matrix proteins play important roles in shell formation by regulating the assembly of organic matrix and minerals. Here, we obtained a new matrix protein (hicraqin) from <em>Hyriopsis cumingii</em>. The amino acid sequence of hicraqin contains multiple aggregated (Gly)n (n > 2) residues, a feature unique to silk-like matrix proteins. <em>In situ</em> hybridization studies and tissue expression patterns demonstrated that hicraqin may be a prismatic layer matrix protein. In vitro experiments were performed using the peptide N-hicraqin (the N-terminal free sequence of hicraqin). In the <em>in vitro</em> crystallization of calcium carbonate, crystals resembling dumbbell, spindle, and lotus aragonite crystals were observed under scanning electron microscopy and confirmed as calcite by Raman spectroscopy. In the <em>in vitro</em> crystallization system of calcium carbonate with the addition of magnesium ions, aragonite plates were generated with 50 μg/mL of the peptide N-hicraqin. The fluorescent labeling analysis indicated that N-hicraqin was involved in the crystallization process. The crystallization rate experiment showed that the peptide N-hicraqin plays a role in promoting crystallization. Following the silencing of the hicraqin gene by RNA interference, its expression was reduced by about 61 %. There was incomplete formation of the organic framework outside the prismatic layer. Overall, the present study showed that N-hicraqin participates in the crystallization process and acts as a framework protein that influences the formation of the organic framework of the prismatic layer.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106642"},"PeriodicalIF":1.4,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142795062","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

Recent trends in production and potential applications of microbial amylases: A comprehensive review 微生物淀粉酶的生产和潜在应用的最新趋势：综述。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-05 DOI: 10.1016/j.pep.2024.106640

Zain Ali , Muhammad Abdullah , Muhammad Talha Yasin , Kinza Amanat , Mohsin Sultan , Aqdas Rahim , Fatima Sarwar

{"title":"Recent trends in production and potential applications of microbial amylases: A comprehensive review","authors":"Zain Ali , Muhammad Abdullah , Muhammad Talha Yasin , Kinza Amanat , Mohsin Sultan , Aqdas Rahim , Fatima Sarwar","doi":"10.1016/j.pep.2024.106640","DOIUrl":"10.1016/j.pep.2024.106640","url":null,"abstract":"<div><div>α-amylases are vital biocatalysts that constitute a billion-dollar industry with a substantial and enduring global demand. Amylases hydrolyze the α-1,4-glycosidic linkages in starch polymers to generate maltose and malto-oligosaccharides subunits. Amylases are key enzymes that have promising applications in various industrial processes ranging from pharmaceutical, pulp and paper, textile food industries to bioremediation and biofuel sectors. Microbial enzymes have been widely used in industrial applications owing to their ease of availability, cost-effectiveness and better stability at extreme temperatures and pH. α-amylases derived from distinct microbial origins exhibit diverse characteristics, which make them suitable for specific applications. The routine application of immobilized enzymes has become a standard practice in the production of numerous industrial products across the pharmaceutical, chemical, and food industries. This review details the structural makeup of microbial α-amylase to understand its thermodynamic characteristics, aiming to identify key areas that could be targeted for improving the thermostability, pH tolerance and catalytic activity of α-amylase through various immobilization techniques or specific enzyme engineering methods. Additionally, the review briefly explores the enzyme production strategies, potential sources of α-amylases, and use of cost-effective and sustainable raw materials for enzyme production to obtain α-amylases with unconventional applications in various industrial sectors. Major hurdles, challenges and future prospects involving microbial α-amylases has been briefly discussed by considering its diverse applications in industrial bioprocessing.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106640"},"PeriodicalIF":1.4,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142792164","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 development of nanobody-based affinity chromatography for AAV8 purification 基于纳米体亲和色谱法纯化AAV8的高效研究。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-03 DOI: 10.1016/j.pep.2024.106638

Guanghui Li , Xiaofei Li , Min Zhu, Peng Qiao, Weiwei Ji, Yuping Huang, Yicai Zhang, Xuee Li, Yakun Wan

{"title":"Efficient development of nanobody-based affinity chromatography for AAV8 purification","authors":"Guanghui Li , Xiaofei Li , Min Zhu, Peng Qiao, Weiwei Ji, Yuping Huang, Yicai Zhang, Xuee Li, Yakun Wan","doi":"10.1016/j.pep.2024.106638","DOIUrl":"10.1016/j.pep.2024.106638","url":null,"abstract":"<div><div>Adeno-associated virus serotype 8 (AAV8) is a highly effective vector for gene therapy. However, its purification remains challenging due to its low natural abundance and stringent purity requirements. This study aimed to develop an affinity chromatography resin utilizing nanobodies (Nbs) to enhance AAV8 purification efficiency. An AAV8-specific Nb library was constructed, leading to the identification of Nb9 as the most promising candidate based on its high binding affinity, stability and yield. Nb9 was expressed in <em>Pichia pastoris</em>, resulting in high yield and exceptional purity. Two types of agarose resins, Epoxy activated Bestarose 6B and PabPur SulfoLink Beads 4FF, were employed for Nb9 conjugation. Epoxy activated Bestarose 6B resin exhibited a significantly higher ligand density (9.12 mg/mL). Binding capacity assessments of the LQ01 resin demonstrated optimal performance at pH 7.0, with diminishing efficacy at lower and higher pH levels. Different NaCl concentrations influenced the binding efficiency, providing critical insights for refining purification conditions. Purification trials exhibited high specificity, purity and consistent VP protein ratio, as evidenced by SDS-PAGE analysis, confirming effective AAV8 capture and elution. Furthermore, the resin demonstrated robust performance across repeated cycles, retaining 71.9 % of its initial binding capacity after 20 uses and maintaining stability with only a 6 % reduction after 7 days at 37 °C. These findings highlight LQ01's potential for scalable and cost-effective AAV8 purification, while demonstrating the broader applicability of Nbs in affinity chromatography and biotechnological processes.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106638"},"PeriodicalIF":1.4,"publicationDate":"2024-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142786777","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

Expression of the Fusarium graminearum galactose oxidase GaoA in Saccharomyces cerevisiae 谷草镰刀菌半乳糖氧化酶gaa在酿酒酵母中的表达。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-11-29 DOI: 10.1016/j.pep.2024.106637

Lucas Yudai Nozaki, Nathalia Rodrigues Bulka, Karina Lima dos Reis, Damaris Batistão Martim, Fausto Fernandes de Castro, Ione Parra Barbosa-Tessmann

{"title":"Expression of the Fusarium graminearum galactose oxidase GaoA in Saccharomyces cerevisiae","authors":"Lucas Yudai Nozaki, Nathalia Rodrigues Bulka, Karina Lima dos Reis, Damaris Batistão Martim, Fausto Fernandes de Castro, Ione Parra Barbosa-Tessmann","doi":"10.1016/j.pep.2024.106637","DOIUrl":"10.1016/j.pep.2024.106637","url":null,"abstract":"<div><div>Galactose oxidase, produced by fungi of the genus <em>Fusarium</em>, is an enzyme of great biotechnological importance. The <em>gaoA</em> gene has been recombinantly expressed in several hosts but has yet to be in <em>Saccharomyces cerevisiae</em>. This work aimed to express the <em>Fusarium graminearum</em> GaoA enzyme in <em>S. cerevisiae</em>. The full-length and the truncated <em>F. graminearum gaoA</em> gene were subcloned into a yeast expression vector. The GaoA enzyme expression level in <em>S. cerevisiae</em> was higher when the truncated gene, which codes for the mature form of the enzyme, was used. After purification of the expressed enzyme on a Sepharose® 6B column, the obtained yield of the pure and active enzyme was 16.7 mg/L. The purified protein showed a <em>K</em><sub>M</sub> of 9.8 mM, lower than that of the wild-type enzyme, and a <em>k</em><sub>cat</sub>/<em>K</em><sub>M</sub> of 2.9 × 10<sup>7</sup> M<sup>−1</sup>s<sup>−1</sup>, higher than that of the wild-type enzyme. The expressed recombinant protein used several common substrates for galactose oxidase, such as galactose, raffinose, and 1,3-dihydroxyacetone dimer. In addition, it had increased activity on guar gum, lactose, and Arabic gum compared with the wild-type enzyme. The obtained enzyme's characteristics are compatible with the galactose oxidase biotechnological applications.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106637"},"PeriodicalIF":1.4,"publicationDate":"2024-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142771648","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

The combined effect of the gene copy number and chaperone overexpression on the recombinant bovine chymosin production in Pichia pastoris, with mutant ADH2 promoter 基因拷贝数和伴侣蛋白过表达对ADH2启动子突变的毕赤酵母重组牛凝乳酶产生的联合影响。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-11-29 DOI: 10.1016/j.pep.2024.106636

Fatma Ersöz , Mehmet İnan

{"title":"The combined effect of the gene copy number and chaperone overexpression on the recombinant bovine chymosin production in Pichia pastoris, with mutant ADH2 promoter","authors":"Fatma Ersöz , Mehmet İnan","doi":"10.1016/j.pep.2024.106636","DOIUrl":"10.1016/j.pep.2024.106636","url":null,"abstract":"<div><div>Chymosin is an enzyme used to coagulate milk, in the cheese industry. This study aimed to increase recombinant production of the chymosin in <em>Pichia pastoris</em> by determining the optimum copy number and overproduction of a Protein Disulfide Isomerase (<em>PpPDI)</em> chaperon protein. <em>Bos taurus</em> chymosin was expressed under the control of a mutant <em>ADH2</em> promoter. The clones containing 1–4 gene copy numbers of the chymosin were constructed using the <em>in vitro</em> cloning method, and the effect of chaperone protein on chymosin secretion was investigated.</div><div>The enzyme production levels are 4, 6.3, 4.5, and 3 IMCU/mL for 1, 2, 3, and 4-copy clones. The secreted chymosin levels increased up to two copies, and increasing the number of copies decreased the secretion level. Therefore, <em>PpPDI</em> was over-expressed in the clones regulated with the <em>ADH2</em> promoter. The over-expression of <em>PDI</em> gene increased chymosin secretion in clones compared to the counterpart host. However, the highest chymosin level was obtained with C2 (2-copy chymosin containing clone; 6.3 IMCU/mL) and C2P2 (2-copy chymosin/2-copy PDI containing clone; 8.2 IMCU/mL).</div><div>The maximum production was 39 IMCU/mL with the clone C2P2 in the fermenter scale production. The enzyme activity increased approximately 2-fold by adding two copies of the chaperone protein. The combined effect of gene copy number and chaperone overexpression on chymosin production was investigated. Two copies of the chymosin and <em>PpPDI</em> genes were the optimum among the tested clones.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106636"},"PeriodicalIF":1.4,"publicationDate":"2024-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142771663","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 purification and excitation energy transfer characterization of phycoerythrin 545 from Rhodomonas sp. Rhodomonas藻红蛋白545的高效纯化及激发能转移特性研究

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-11-26 DOI: 10.1016/j.pep.2024.106634

Yang Pu , Shuo Dong , Jiayu Wang , Min Li , Kai Dong , Wenjun Li , Zhihong Tang

{"title":"Efficient purification and excitation energy transfer characterization of phycoerythrin 545 from Rhodomonas sp.","authors":"Yang Pu , Shuo Dong , Jiayu Wang , Min Li , Kai Dong , Wenjun Li , Zhihong Tang","doi":"10.1016/j.pep.2024.106634","DOIUrl":"10.1016/j.pep.2024.106634","url":null,"abstract":"<div><div>Cryptomonad phycoerythrin 545 (PE545) is an important type of phycobiliprotein in basic research and technological innovations. Herein, we report a minimalistic hydrophobic chromatography method for its purification. High purity was achieved, with a purity ratio (<em>A</em><sub>545</sub>/<em>A</em><sub>280</sub>) of 13.66 and a recovery ratio of 78.63 %. Following SDS-PAGE, Coomassie Brilliant Blue staining revealed three bands at 9 kDa, 10 kDa, and 20 kDa, corresponding to α<sub>1</sub>, α<sub>2</sub> and β subunits. Multiple spectral characteristics were analyzed to ensure that optical activity was consistent with that of the natural protein. Absorption and fluorescence spectroscopies of purified PE545 displayed a strong absorption peak at 545 nm, a shoulder peak at 564 nm, and a fluorescence emission peak at 587 nm, which confirmed unchanged energy transfer properties. Furthermore, the structural and functional integrity, especially the existence of strongly coupled central chromophore pairs with excitation delocalization, was verified by circular dichroism and ultrafast absorption spectroscopy. From the studies of ultrafast absorption spectroscopy of excitation energy transfer (EET) of PE545, four decay components with lifetimes at 0.5 ps, 2.2 ps, 63 ps, and 3000 ps were obtained. In addition, the dynamics of these components confirmed the EET pathways from the central PEB chromophore pairs to the peripheral pigments and localized in the lowest state. Our work will be of considerable value for both fundamental research and applications of PE545.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106634"},"PeriodicalIF":1.4,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142751389","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

Isolation and crystallization of copper resistance protein B (CopB) from Acinetobacter baumannii 鲍曼不动杆菌铜抗性蛋白B （CopB）的分离与结晶

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-11-26 DOI: 10.1016/j.pep.2024.106635

Niloofar Nayeri , Kamil Górecki , Karin Lindkvist-Petersson , Pontus Gourdon , Ping Li

{"title":"Isolation and crystallization of copper resistance protein B (CopB) from Acinetobacter baumannii","authors":"Niloofar Nayeri , Kamil Górecki , Karin Lindkvist-Petersson , Pontus Gourdon , Ping Li","doi":"10.1016/j.pep.2024.106635","DOIUrl":"10.1016/j.pep.2024.106635","url":null,"abstract":"<div><div><em>Acinetobacter baumannii</em> (<em>A. baumannii</em>) is an opportunistic, Gram-negative human pathogen, which is predominantly found in hospital patients. Its antimicrobial resistance is escalating, leading to less efficient treatments, and an increasing interest in identifying new therapeutic drugs. Metals as antimicrobials are vital in healthcare and agriculture, and copper-containing surfaces are known to reduce microbial counts, also in clinical settings. Indeed, copper (Cu) is an essential element required for survival in all organisms from bacteria to humans, but nevertheless elevated levels are highly toxic for cells. Through different regulatory mechanisms, cells maintain Cu homeostasis, and ion channels and transporters are critical in this process. Precise understanding of such ion transport requires insight into the protein structures of the involved proteins, which will also provide information important for applied sciences. Considering the medical significance of <em>A. baumannii</em> and the possibility to exploit Cu to handle such infections, channels and transporters represent appealing targets. Here we approached the putative outer membrane CopB (Copper resistance protein B) from <em>A. baumannii</em> that is postulated to conduct Cu, with characterization of its structure and function as well as to enable rational drug-design. To this end, we demonstrate in this work procedures to produce purified sample and to recover diffracting protein crystals of CopB. The protein was overproduced in <em>E. coli</em> and membrane extracted in a range of detergents. The solubilized protein was subjected to crystallization, which yielded hits that scatter X-rays to low resolution. Our findings have the potential to pave the way for subsequent drug discovery.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106635"},"PeriodicalIF":1.4,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142747969","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