Analytical biochemistry最新文献_第8页

Metabolomics approach using HR-MAS NMR spectroscopy for the assessment of metabolic profiles of uterine fibroids 代谢组学方法使用HR-MAS核磁共振光谱评估子宫肌瘤的代谢谱

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-29 DOI: 10.1016/j.ab.2025.115885

Senem Arda Düz , Akın Mumcu , Berat Doğan , Erdinç Sarıdoğan , Görkem Tuncay , Taylan Onat , Abdullah Karaer

{"title":"Metabolomics approach using HR-MAS NMR spectroscopy for the assessment of metabolic profiles of uterine fibroids","authors":"Senem Arda Düz , Akın Mumcu , Berat Doğan , Erdinç Sarıdoğan , Görkem Tuncay , Taylan Onat , Abdullah Karaer","doi":"10.1016/j.ab.2025.115885","DOIUrl":"10.1016/j.ab.2025.115885","url":null,"abstract":"<div><div>The aim of this study is to determine dysregulated metabolites and metabolic pathways in uterine fibroids and in the myometrial tissue from which uterine fibroids are derived. Fifteen (15) patients underwent hysterectomy because of uterine fibroids and 14 controls were included in this study. <sup>1</sup>H HR-MAS NMR spectroscopy data were obtained from uterine fibroid tissue, the adjacent healthy myometrial tissue from cases, and myometrial tissue from controls. PCA and PLS-DA score plots from multivariate statistical analysis of pre-processed spectral data demonstrated a distinction between cases and control groups. The levels of lactate, alanine, glutamate, glutamine, methionine, acetone, isocitrate, choline, glycerophosphocholine, phosphocholine, <em>o</em>-phosphoethanolamine, taurine, myo-inositol, <em>p</em>-methylhistidine, phenylacetate, ascorbate, glucose, and methylhistidine were significantly higher in uterine fibroid tissue compared to the neighboring healthy myometrial tissue. Additionally, when adjacent healthy myometrial tissue was compared to control myometrial tissue, significantly lower levels of valine, leucine, isoleucine, ethanol, arginine, N-acetyl tyrosine, acetone, <em>p</em>-methylhistidine, glucose, phenylacetate, myo-inositol, and alpha-glucose were observed. The study provides a foundational framework by revealing the metabolomic heterogeneity of uterine fibroids. Strategies should be developed to target the metabolic alterations that contribute to the growth of these common tumors.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"704 ","pages":"Article 115885"},"PeriodicalIF":2.6,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143935883","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

nCas9-based method for rolling-circle DNA substrate generation 基于ncas9的滚圈DNA底物生成方法

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-25 DOI: 10.1016/j.ab.2025.115883

Nischal Sharma, Kelsey S. Whinn, Harshad Ghodke, Antoine M. van Oijen, Jacob S. Lewis, Lisanne M. Spenkelink

{"title":"nCas9-based method for rolling-circle DNA substrate generation","authors":"Nischal Sharma, Kelsey S. Whinn, Harshad Ghodke, Antoine M. van Oijen, Jacob S. Lewis, Lisanne M. Spenkelink","doi":"10.1016/j.ab.2025.115883","DOIUrl":"10.1016/j.ab.2025.115883","url":null,"abstract":"<div><div>Rolling-circle DNA replication is a DNA-duplication mechanism whereby circular DNA templates are continuously copied to produce long DNA products. It is widely used in molecular diagnostics, DNA sequencing, nanotechnology, and <em>in vitro</em> DNA replication studies. The efficiency of rolling-circle replication reaction heavily relies on the quality of the rolling-circle DNA template. Existing methods to create rolling-circle DNA substrates often rely on unique restriction sites and have limited control over replication fork topology and position. To address these limitations, we present a straightforward, customizable, and efficient strategy for producing rolling-circle DNA substrates with control over gap size and fork position. Our method relies on the use of nickase Cas9 (nCas9), which can be programmed to target specific DNA sequences using guide RNAs. In a one-pot reaction, we target nCas9 to four sites on an 18-kb plasmid to create 8–11-bp fragments. These fragments are removed and a flap oligo is ligated, to construct a fork with precisely controlled flap length and gap size. We demonstrate the application of this DNA substrate in an <em>in vitro</em> single-molecule rolling-circle DNA-replication assay. With our method, any plasmid DNA can be converted into a rolling-circle template, permitting generation of more physiologically-relevant DNA templates.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115883"},"PeriodicalIF":2.6,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143881540","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

Proximity rolling circle amplification based generation of lighting-up aptamer for sensitive and label-free MicroRNA analysis 基于邻近滚动圆扩增的点亮适体的产生，用于敏感和无标记的MicroRNA分析

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-25 DOI: 10.1016/j.ab.2025.115884

Chaogang Yuan , Lifang Cao , Honglian Shen , Yuexiang Zhang

{"title":"Proximity rolling circle amplification based generation of lighting-up aptamer for sensitive and label-free MicroRNA analysis","authors":"Chaogang Yuan , Lifang Cao , Honglian Shen , Yuexiang Zhang","doi":"10.1016/j.ab.2025.115884","DOIUrl":"10.1016/j.ab.2025.115884","url":null,"abstract":"<div><div>The dysregulated expression of microRNA (miRNA) has been strongly linked to pneumonia. However, ultrasensitive miRNA detection remains technically challenging due to their short sequences and low abundance in biological samples. Herein, we developed a catalytic hairpin assembly -triggered proximity rolling circle transcription system for synthesizing malachite green (MG)-binding RNA aptamers, enabling label-free and highly sensitive detection of miRNA-21. In this system, target miRNA-21 initiates hairpin probe structural rearrangement, promoting miRNA-21 recycling and activating dual rolling circle transcription reactions. The resulting long RNA transcripts undergo partial hybridization, yielding numerous functional MG RNA aptamers. Upon binding to MG dye, these aptamers generate a robust fluorescence signal, allowing ultrasensitive miRNA-21 detection at concentrations as low as 0.51 fM. The assay exhibits high specificity, discriminating miRNA-21 from single-base mismatched sequences, and shows promise for monitoring miRNA-21 expression in clinical samples. By combining the signal amplification of rolling circle transcription with the high-affinity recognition of MG aptamers, this method achieves a low background, superior signal-to-noise ratio, and exceptional sensitivity. Furthermore, the strategy can be readily adapted to detect other trace biomarkers by modifying the target-recognition sequence.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115884"},"PeriodicalIF":2.6,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143894737","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

Development of a competitive ELISA based on Brucella neotomae lipopolysaccharide for detecting brucellosis in livestock 基于新生布鲁氏菌脂多糖的竞争性酶联免疫吸附测定家畜布鲁氏菌病

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-23 DOI: 10.1016/j.ab.2025.115880

Guo-Hua Cai, Chao-Yue Guo, Kai-Xuan Guo, Jian-Dong Zhang, Huan-Chun Chen, Zheng-Fei Liu

{"title":"Development of a competitive ELISA based on Brucella neotomae lipopolysaccharide for detecting brucellosis in livestock","authors":"Guo-Hua Cai, Chao-Yue Guo, Kai-Xuan Guo, Jian-Dong Zhang, Huan-Chun Chen, Zheng-Fei Liu","doi":"10.1016/j.ab.2025.115880","DOIUrl":"10.1016/j.ab.2025.115880","url":null,"abstract":"<div><div>Brucellosis, a global zoonotic threat, requires efficient diagnostic tools for effective surveillance. Commercial competitive enzyme-linked immunosorbent assay (cELISA) predominantly utilizes smooth lipopolysaccharides (S-LPS) extracted from <em>B. abortus</em> and <em>B. melitensis</em> as key antigens for brucellosis serodiagnosis. However, culturing pathogens requires facilities with high biosafety, which is operationally complex and economically demanding. In this study, we developed a cELISA using LPS extracted from <em>B. neotomae</em>, which can be handled more facilely in biosafety level 2 conditions, and analyzed clinical adaptability of the cELISA. The optimized cELISA demonstrated lower detection limits, which was 2–4 times more analytically sensitive than commercial kit by detecting sera against <em>B. melitensis</em> and <em>B. abortus</em>. No cross-reactivity was observed with sera infected with other bacteria, including <em>E. coli</em>, <em>Salmonella</em>, <em>Y. enterocolitica</em>, and <em>M. tuberculosis</em>. The diagnostic sensitivity and specificity of the cELISA were 100 % (40/40) and 100 % (40/40), respectively. The coefficients of variation were less than 10 %. Moreover, compared to the commercial kit, the developed ELISA achieved agreement of 92.51 % across 427 sera from vaccinated livestock, and agreement of 96.98 % across 696 sera from non-vaccinated livestock. In conclusion, the cELISA exhibits excellent sensitivity, specificity and repeatability, indicating its potential for brucellosis diagnosis in livestock.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115880"},"PeriodicalIF":2.6,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143877194","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

Real-time monitoring method of microbial growth using a simple pressure-based respiration detection system 利用简单的压力呼吸检测系统实时监测微生物生长的方法

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-22 DOI: 10.1016/j.ab.2025.115879

Nara Shin , Jinok Oh , Yebin Han , Gaeun Lim , Jeong Chan Joo , Woo-Young Jeon , Jungoh Ahn , Hee Taek Kim , Shashi Kant Bhatia , Yung-Hun Yang

{"title":"Real-time monitoring method of microbial growth using a simple pressure-based respiration detection system","authors":"Nara Shin , Jinok Oh , Yebin Han , Gaeun Lim , Jeong Chan Joo , Woo-Young Jeon , Jungoh Ahn , Hee Taek Kim , Shashi Kant Bhatia , Yung-Hun Yang","doi":"10.1016/j.ab.2025.115879","DOIUrl":"10.1016/j.ab.2025.115879","url":null,"abstract":"<div><div>Dry cell weight (DCW) and optical density (OD) measurement methods provide useful data for assessing microbial growth. However, their sampling process is labor-intensive and time-consuming. Therefore, we aimed to evaluate a method for measuring microbial growth through continuous CO<sub>2</sub> measurement under aerobic conditions using a pressure-based respiration detection system, which is traditionally used in anaerobic environments and applies measurement of reduced pressure by capturing CO<sub>2</sub> with KOH. The pressure reduction rate, OD, and DCW values were compared during <em>Ralstonia eutropha</em> H16 culture, which revealed a correlation of R<sup>2</sup> of 0.99 between the pressure reduction and DCW and a change of DCW (g/L) per pressure (1 mbar) of −0.02 g/L. It showed theoretical limit of detection at 14.67 mbar corresponding to 0.0428 g/L of DCW and theoretical limit of quantification at 48.9 mbar as lower limits. When the pressure-based method was applied to compare carbon source utilization and growth of different strains, such as <em>E. coli</em> sp., <em>Pseudomonas</em> sp., <em>Burkholderia</em> sp., and <em>Bacillus</em> sp., it showed a high correlation with DCW. Overall, these results demonstrate that the pressure-based respiration detection system is a reliable tool for microbial growth monitoring and offers significant advantages by providing real-time data with less labor.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115879"},"PeriodicalIF":2.6,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143869448","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

Development of a high-sensitivity double kinetic assay for creatinine using the enzymatic cycling method and its application to serum samples 酶循环法测定肌酐高灵敏度双动力学分析方法的建立及其在血清样品中的应用

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-21 DOI: 10.1016/j.ab.2025.115877

Eisaku Hokazono , Yasunori Haraguchi , Ai Higashinakao, Akito Mominoki, Takeshi Uchiumi, Yuzo Kayamori

{"title":"Development of a high-sensitivity double kinetic assay for creatinine using the enzymatic cycling method and its application to serum samples","authors":"Eisaku Hokazono , Yasunori Haraguchi , Ai Higashinakao, Akito Mominoki, Takeshi Uchiumi, Yuzo Kayamori","doi":"10.1016/j.ab.2025.115877","DOIUrl":"10.1016/j.ab.2025.115877","url":null,"abstract":"<div><div>Estimated glomerular filtration rate (eGFR) is often used as a measure of renal function in clinical practice owing to its simplicity. Serum creatinine levels are essential for eGFR calculation. Although the Jaffé method is widely used for creatinine measurement, it exhibits low specificity and sensitivity. Development of various enzyme methods has increased its specificity; however, its sensitivity is still insufficient for accurate eGFR calculation. To overcome this issue, we developed a highly sensitive assay for creatinine detection using enzymatic cycling reaction in this study. Our method consisted of two steps: Endogenous ammonia elimination and creatinine analysis. NADH derived from creatinine changed the water-soluble tetrazolium salt-8 color in the presence of the electron carrier, 1-methoxy-5-methylphenazinium methylsulfate. The cycling oxidation–reduction of 1-methoxy-5-methylphenazinium methylsulfate to NADH facilitated the coloration of water-soluble tetrazolium salt-8, aiding in creatinine measurement with high sensitivity. The within-run reproducibility of the developed method was good (<1.76 % at each concentration tested), with a detection limit of 1.00 μmol/L, making it approximately 9 times more sensitive than the Jaffé method. Notably, its correlation with the high-performance liquid chromatography method was excellent (<em>r</em> = 0.976). Overall, this study successfully developed a new, rapid, simple, and highly sensitive method for creatinine analysis.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115877"},"PeriodicalIF":2.6,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143906673","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

Highly-sensitive peptide array using peptides immobilized on microbeads: Application to cow's milk allergy analysis 微珠固定多肽的高敏感多肽阵列：在牛奶过敏分析中的应用

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-19 DOI: 10.1016/j.ab.2025.115865

Hideo Tashiro , Tomoko Tashiro , Fumiya Yamaide , Taiji Nakano , Yuzaburo Inoue , Yuki Takase , Erika Sawano , Naoki Shimojo

{"title":"Highly-sensitive peptide array using peptides immobilized on microbeads: Application to cow's milk allergy analysis","authors":"Hideo Tashiro , Tomoko Tashiro , Fumiya Yamaide , Taiji Nakano , Yuzaburo Inoue , Yuki Takase , Erika Sawano , Naoki Shimojo","doi":"10.1016/j.ab.2025.115865","DOIUrl":"10.1016/j.ab.2025.115865","url":null,"abstract":"<div><h3>Analysis</h3><div>with peptide microarrays containing linear epitopes of allergenic proteins is expected to provide information on the clinical status of the patient, but peptide arrays are still limited to research use. We thus aimed at developing a simple and sensitive peptide array that operates with more cost-effective ECL detection, so that it can be routinely used in clinical practice.</div><div>For this purpose, instead of directly immobilizing the peptides onto the microarray surface as in the previous reports, we developed a two-step immobilization technique using microbeads. Peptides biotinylated at the N-terminal are first bound to microbeads with streptavidin (sAV) on the surface, followed by immobilization of the peptide-bound beads onto the microarray substrate using the photoreactive crosslinker. In this way, we were able to overcome the limitations of direct immobilization in increasing the amount and accessibility of peptides and greatly enhance the sensitivity so that ECL detection became possible. In the present study, we analyzed sera from cow's milk allergy (CMA) patients with our peptide array containing 20 peptides from αS1-casein. The results showed that IgE epitope patterns of patients could be visualized individually, and confirmed that the pattern is unique to each patient.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115865"},"PeriodicalIF":2.6,"publicationDate":"2025-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143878629","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

Examining cortisol ELISA in dried matrix spots: implications for analyte measurement and stability 检测皮质醇ELISA在干燥基质斑点：对分析物的测量和稳定性的影响

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-19 DOI: 10.1016/j.ab.2025.115878

Jeanne V. Samsonova , Nikolay Yu. Saushkin , Aleksei K. Piskunov

{"title":"Examining cortisol ELISA in dried matrix spots: implications for analyte measurement and stability","authors":"Jeanne V. Samsonova , Nikolay Yu. Saushkin , Aleksei K. Piskunov","doi":"10.1016/j.ab.2025.115878","DOIUrl":"10.1016/j.ab.2025.115878","url":null,"abstract":"<div><div>Whole blood, plasma, whole milk and urine samples from domestic goats (<em>Capra hircus</em>) in both liquid and dried form were analysed by quantitative ELISA. Dried matrix samples were prepared using two types of absorbing support: glass fibre strip (strip-dried samples) and cellulose membrane (dried spots). Strip-dried samples have advantages over dried spots due to better cortisol recovery, reproducibility, ease of dried sample aliquoting and no need for recalculations against standard protocol. In the cortisol quantitative ELISA, the best concordance between the results was achieved in the pair “strip-dried whole blood/native plasma” and “strip-dried urine/native urine”. The correlation coefficient of cortisol results in strip-dried blood, plasma and urine vs liquid samples ranged from 0.90 to 0.97 (p < 0.001). The mean recovery of cortisol from blood and plasma samples spotted on cellulose across a wide range of concentrations was much lower that from strip-dried blood and plasma, and varied between 40 and 60 %. Dried plasma samples with high cortisol content (>400 nmol/L) showed a slight decrease in the recovered cortisol concentrations on 20 % average. Cortisol in dried blood, plasma and milk samples was stable over a period of six months at 4 °C, a week at 37 °C or 24 h at 60 °C.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115878"},"PeriodicalIF":2.6,"publicationDate":"2025-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143859006","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

Mechanism of DNA multimerization caused by strand-displacement DNA polymerases 链置换DNA聚合酶引起DNA聚合的机制

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-19 DOI: 10.1016/j.ab.2025.115876

Assol R. Sakhabutdinova, Ravil R. Garafutdinov

{"title":"Mechanism of DNA multimerization caused by strand-displacement DNA polymerases","authors":"Assol R. Sakhabutdinova, Ravil R. Garafutdinov","doi":"10.1016/j.ab.2025.115876","DOIUrl":"10.1016/j.ab.2025.115876","url":null,"abstract":"<div><div>It has been recently shown that for Bst DNA polymerase, the side isothermal amplification reaction named multimerization (MM) proceeds under certain conditions. MM hinders interpretation of amplification results and reduces the accuracy and reliability of DNA/RNA diagnostics. Here, the mechanism of MM caused by strand-displacement DNA polymerases is reported. The mechanism includes the following key stages: 1) envelopment of the enzyme globule by the synthesized DNA strand, facilitated by DNA breathing, 2) convergence of the 3′-ends of the DNA strands and pseudo-cyclic trigger DNA structure formation, 3) synthesis of the products with repeated motifs resulting in their expansion due to DNA slippage. Initiation of MM reaction occurs with extremely low probability, however, the resulting few trigger DNA structures are efficiently amplified and ultimately lead to the accumulation of nonspecific amplicons (multimers). Molecular models with certain steric and thermodynamic characteristics were used to confirm the proposed mechanism. The highest MM efficiency was observed for DNA templates and reaction conditions that facilitated DNA breathing, complete envelopment of the enzyme globule with DNA strands and convergence of their 3′-ends.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115876"},"PeriodicalIF":2.6,"publicationDate":"2025-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143850823","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 functional properties of highly-purified N-terminal domain of human NaPi2b by scalable “resin overload” technique 用可扩展的“树脂过载”技术分离纯化人类NaPi2b蛋白n端结构域及其功能特性

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-18 DOI: 10.1016/j.ab.2025.115875

Daria Savenkova , Irina Makarenko , Daria Nedorezova , Ramziya Kiyamova , Mikhail Bogdanov

{"title":"Isolation and functional properties of highly-purified N-terminal domain of human NaPi2b by scalable “resin overload” technique","authors":"Daria Savenkova , Irina Makarenko , Daria Nedorezova , Ramziya Kiyamova , Mikhail Bogdanov","doi":"10.1016/j.ab.2025.115875","DOIUrl":"10.1016/j.ab.2025.115875","url":null,"abstract":"<div><div>Despite the enthusiasm and advances in the purification of native and engineered full-length membrane proteins, little attention has been paid to their fragments which could serve as attractive inspiration for function, regulation, or targeting of full-length membrane protein with therapeutic antibodies (Abs). Production of recombinant fragments of “therapeutic” membrane proteins for early-stage discovery research requires their purification to near homogeneity. It is important not only for the production of biotherapeutic antibodies but also for structural and functional studies of competitive protein-Abs, protein-protein, and lipid-protein interactions which heavily rely on the purity and quality of the isolated protein fragment of interest. The development of novel strategies for simple but still highly efficient protein purification remains a one of main research focus in the biotechnology and biomedicine because conventional purification approaches require complex manipulation steps and are timely and costly. Here, we would like to introduce a simple and rapid protein purification strategy for the human NaPi2b N-terminal (NT) sequence recombinantly expressed in a bacterial host at a laboratory scale. We demonstrate that “resin overload” e.g. the conditions when loading exceeds dynamic binding capacity can be counterintuitively but intelligently utilized to isolate highly purified protein fragments and prevent non-specific low-affinity binding of contaminant endogenous host proteins. The results showed that this method allowed us to achieve the highest purity while maintaining both immunogenic (recognition by Abs) and functional (phosphorylation) properties of the NaPi2b NT sequence. Although adaptations are required on a case-to-case basis, we believe this work can inspire other researchers working with the purification of protein and protein fragments to apply this proof-of-principle in a scalable manner.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115875"},"PeriodicalIF":2.6,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143850821","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