Protein expression and purification最新文献

Development of a novel capture step for purification of antigen binding fragments (Fabs).

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-17 DOI: 10.1016/j.pep.2024.106647

Anupa Anupa, Anurag S Rathore

引用次数: 0

A simple freeze-thaw based method for efficient purification of recombinant human proinsulin from inclusion bodies. 从包涵体中高效纯化重组人胰岛素的简单冻融法。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-15 DOI: 10.1016/j.pep.2024.106645

S Rajesh, Swaraj Jathar, Reema Banarjee, Monika Sharma, Shivani Palkar, S Shiva Shankar, Mahesh J Kulkarni

{"title":"A simple freeze-thaw based method for efficient purification of recombinant human proinsulin from inclusion bodies.","authors":"S Rajesh, Swaraj Jathar, Reema Banarjee, Monika Sharma, Shivani Palkar, S Shiva Shankar, Mahesh J Kulkarni","doi":"10.1016/j.pep.2024.106645","DOIUrl":"10.1016/j.pep.2024.106645","url":null,"abstract":"Insulin is a pivotal peptide hormone essential for regulating glucose homeostasis. It has been known for over 100 years, but its production and purification methods are still under improvement. Escherichia coli based bacterial expression system is primarily used for insulin production. The human insulin protein expressed in bacteria usually forms inclusion bodies, complicating the purification process. Traditionally, insulin purification is a time-consuming process involving urea-based denaturation methods, and various refolding techniques, followed by extensive chromatographic methods. Here, we report an easy and efficient purification of human proinsulin involving freeze-thaw based solubilization method. The extracted proinsulin inclusion bodies are treated with different concentrations of urea, followed by a freeze-thaw based solubilization. The freezing was carried out at various temperatures, mainly -80 °C, -20 °C, and -196 °C to determine the optimum condition for solubilization. Highest solubilization of proinsulin from the inclusion body was achieved with 0.5M urea and -20 °C. Further Nickel NTA-based purification was performed, and the purified protein was characterized for disulfide mapping by high-resolution mass spectrometer (HRMS). We also performed glucose uptake assays to validate the functional properties of purified proinsulin. This freeze-thaw based mild solubilization approach is a fast and effective method for getting bioactive proinsulin, which will help further design better purification and processing strategies for insulin production.","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106645"},"PeriodicalIF":1.4,"publicationDate":"2024-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142838929","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

Improvements in large-scale production of Tobacco Etch Virus protease. 烟草蚀刻病毒蛋白酶大规模生产的改进。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-14 DOI: 10.1016/j.pep.2024.106648

Simon Messing, Kirsten Barnhart, Matthew Drew, Natalie Granato-Guerrero, Carissa Grose, Brianna Higgins, Min Hong, Jenna Hull, Shelley Perkins, Ivy Poon, Nitya Ramakrishnan, Amanda Seabolt, Troy Taylor, Vanessa E Wall, Nicholas Wright, William Gillette, Dominic Esposito

引用次数: 0

MabSelect VH3 Protein A affinity resin effectively separates antibody species containing different numbers of VH3 domain and shows improved aggregate separation capability. MabSelect VH3 蛋白 A 亲和树脂能有效分离含有不同数量 VH3 结构域的抗体种类，并显示出更强的聚集分离能力。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-12 DOI: 10.1016/j.pep.2024.106646

Wanyuan Dong, Rongrong Wang, Yifeng Li

{"title":"MabSelect VH3 Protein A affinity resin effectively separates antibody species containing different numbers of VH3 domain and shows improved aggregate separation capability.","authors":"Wanyuan Dong, Rongrong Wang, Yifeng Li","doi":"10.1016/j.pep.2024.106646","DOIUrl":"10.1016/j.pep.2024.106646","url":null,"abstract":"MabSelect VH3 is a new Protein A resin recently launched by Cytiva. According to the manufacturer, the Protein A ligand of MabSelect VH3 has been engineered to disrupt and reinforce its Fc and VH3 binding capabilities, respectively. Thus, different from regular Protein A resins, this new Protein A resin has affinity for VH3 domain only. The vendor has suggested that MabSelect VH3, owing to its unique selectivity, can separate byproducts that are different from the product in the number of VH3 domain. In the current work, with two concrete cases, we demonstrated that MabSelect VH3 indeed allows effective separation of species containing different numbers of VH3 domain. In addition, we showed that, in comparison to regular Protein A resins, MabSelect VH3 also exhibits improved aggregate separation potential. Thus, for cases where product and byproduct differ in the number of VH3 domain and/or culture harvest contains high percentage of aggregates, MabSelect VH3 is a better alternative than regular Protein A for product capture as it allows simultaneous removal of byproducts and aggregates.","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106646"},"PeriodicalIF":1.4,"publicationDate":"2024-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142823762","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

Heterologous expression, purification and generation of specific antibodies for Annexin A8. 异源表达、纯化和生成 Annexin A8 的特异性抗体。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-12 DOI: 10.1016/j.pep.2024.106644

Zexin Lin, Wei Sun, Xuemei Zhao, Yang Chen, Kerry M Loomes, Hongbo Li, Hannah Xiaoyan Hui, Donghai Wu

{"title":"Heterologous expression, purification and generation of specific antibodies for Annexin A8.","authors":"Zexin Lin, Wei Sun, Xuemei Zhao, Yang Chen, Kerry M Loomes, Hongbo Li, Hannah Xiaoyan Hui, Donghai Wu","doi":"10.1016/j.pep.2024.106644","DOIUrl":"10.1016/j.pep.2024.106644","url":null,"abstract":"Annexin A8 (ANXA8) is a member of the Annexin gene family, with high levels of its mRNA and protein observed in various tumor tissues, making it a potential tumor biomarker. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) to specifically detect the presence and accurately determine the concentration of ANXA8. To this end, we expressed a series of proteins using both Pichia pastoris and Escherichia coli expression systems. Particularly, human ANXA8 expressed in Pichia pastoris was used as the antigen and for antibody screening, while human ANXA2 and ANXA5 expressed in Pichia pastoris and murine ANXA8 expressed in E. coli BL21 (DE3) were used as counter screens to eliminate cross-reactive antibodies and to select antibodies that show specificity to ANXA8 from both human and mouse. Through research efforts with production and purification of recombinant proteins, immunization, specificity screening and selection of epitope pairing, two specific monoclonal antibodies, E9 and B7, apparently targeting different epitopes of ANXA8, were obtained. The specificity of the antibody pair was checked against recombinant human ANXA2 and ANXA5 with ANXA8 as the positive control. Western Blot and Sandwich ELISA demonstrate superb sensitivity and specificity with a detection limit of about 0.065 ng/mL for the latter. In summary, this study provides an experimental tool for the quantitative determination of ANXA8 concentration and a potential tumor biomarker to monitor disease progression.","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106644"},"PeriodicalIF":1.4,"publicationDate":"2024-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142823761","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

Optimized vector for functional expression of the human bitter taste receptor TAS2R14 in HEK293 cells.

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":"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.","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"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":"","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.

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":"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 Lysinibacillus sphaericus Q001. This bacterium was isolated from Pakistan and characterized through various biochemical tests. It was identified as Lysinibacillus sphaericus 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 L. sphaericus 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 L. sphaericus Q001 was toxic against Aedes aegypti larvae with LC50 value of 11 μg/ml. This shows its potential to be used as an alternative to chemical larvicides.","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"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":"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-sn-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.","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"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":"","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.

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":"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 Hyriopsis cumingii. The amino acid sequence of hicraqin contains multiple aggregated (Gly)n (n > 2) residues, a feature unique to silk-like matrix proteins. In situ 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 in vitro 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 in vitro 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.","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"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.

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":"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.","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"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