Protein expression and purification最新文献

Expression and biochemical characterization of a novel NAD⁺-dependent xylitol dehydrogenase from the plant endophytic fungus Trichoderma gamsii.

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-02-04 DOI: 10.1016/j.pep.2025.106687

Shuping Fei, Wenxiu Hu, Jingwen Shu, Ruirui Zhao, Jiatong Zhao, Mengwei Jiang, Wenwen Wu, Chaoqun Lian, Wanggang Tang

{"title":"Expression and biochemical characterization of a novel NAD<sup>+</sup>-dependent xylitol dehydrogenase from the plant endophytic fungus Trichoderma gamsii.","authors":"Shuping Fei, Wenxiu Hu, Jingwen Shu, Ruirui Zhao, Jiatong Zhao, Mengwei Jiang, Wenwen Wu, Chaoqun Lian, Wanggang Tang","doi":"10.1016/j.pep.2025.106687","DOIUrl":"https://doi.org/10.1016/j.pep.2025.106687","url":null,"abstract":"<p><p>Xylitol dehydrogenase (XDH; EC 1.1.1.9), encoded by the XYL2 gene, is a key enzyme in the fungal xylose metabolic pathway. In this work, a putative XDH from the plant endophytic fungus Trichoderma gamsii (TgXDH) was hetero-expressed in Escherichia coli BL21(DE3), purified to the homogeneity, and biochemically characterized. Sequence analysis revealed that TgXDH is 363 amino acids long and belongs to the zinc-containing medium-chain alcohol dehydrogenase superfamily. The size-exclusion chromatography analysis and SDS-PAGE showed that the purified recombinant TgXDH had a native molecular mass of ∼155 kDa and was composed of four identical subunits of molecular mass of ∼39 kDa. The optimum temperature and pH of this enzyme were 25 °C and pH 9.5, respectively. Kinetic analysis showed that it is an NAD<sup>+</sup>-dependent enzyme that has a polyol substrate preference (based on k<sub>cat</sub>/K<sub>m</sub>) in the order xylitol > ribitol ≈ d-sorbitol. The K<sub>m</sub> values for NAD<sup>+</sup> with these three polyols ranged from 0.23 to 0.70 mM. Moreover, TgXDH showed high substrate affinities as compared to most of its homologs. The K<sub>m</sub> values for xylitol, ribitol, and d-sorbitol were 5.23 ± 0.68 mM, 8.01 ± 1.22 mM, and 12.34 ± 1.37 mM, respectively. Collectively, the results will contribute to understanding the biochemical properties of a novel XDH from the filamentous fungi and provide a promising XDH for industrial production of ethanol.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106687"},"PeriodicalIF":1.4,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143365722","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

Optimization on cell lysis and capture process of human adenovirus type 5 produced in suspension HEK293 cells

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-02-04 DOI: 10.1016/j.pep.2025.106686

Sha Yi, Xiaoxu Gu, Youping Jin, Fei Wang, Junjun Jiang

{"title":"Optimization on cell lysis and capture process of human adenovirus type 5 produced in suspension HEK293 cells","authors":"Sha Yi, Xiaoxu Gu, Youping Jin, Fei Wang, Junjun Jiang","doi":"10.1016/j.pep.2025.106686","DOIUrl":"10.1016/j.pep.2025.106686","url":null,"abstract":"<div><div>Adenovirus (Adv) is increasingly recognized for its significance in the fields of gene therapy and viral vector vaccines. The diverse applications in clinical trials and fundamental research necessitate the development of environmentally and economically sustainable purification processes that are straightforward and scalable for both academic and industrial contexts. In the initial segment of this study, we evaluated the lysis efficiency of polysorbate 20 (PS20) in comparison to polysorbate 80 (PS80). Our findings indicated that the viability HEK293 could be reduced to approximately 13 %, with a detectable Adv5 concentration of average 1.62 × 10<sup>9</sup> ifu/mL in the supernatant after an incubation in 1.0 % (w/w) PS20 or PS80 buffer. In the subsequent portion of this research, we employed a high-throughput static binding capacity (SBC) screening tool in conjunction with on-column dynamic binding capacity (DBC) validation to concurrently assess the binding capacity of Adv5 on nine different types of anion exchange media. The results demonstrated that both Sartobind Q membrane and POROS 50HQ resin exhibited binding capacities exceeding 1.0 × 10<sup>13</sup> vp/mL under the testing conditions.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106686"},"PeriodicalIF":1.4,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143348494","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, expression, and characterization of collagen galactosyltransferases from human, sponge, and sea walnut

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-02-02 DOI: 10.1016/j.pep.2025.106685

Jeong Seon Kim, Tingfei Chen, Botao Zhang, Tristin M. Miller, Marisa E. Gilliam, Houfu Guo

{"title":"Cloning, expression, and characterization of collagen galactosyltransferases from human, sponge, and sea walnut","authors":"Jeong Seon Kim, Tingfei Chen, Botao Zhang, Tristin M. Miller, Marisa E. Gilliam, Houfu Guo","doi":"10.1016/j.pep.2025.106685","DOIUrl":"10.1016/j.pep.2025.106685","url":null,"abstract":"<div><div>Collagen is an extracellular matrix protein conserved across animals and viruses, with its function regulated by post-translational modifications of lysine residues. Specifically, certain lysine residues in collagen are hydroxylated to form hydroxylysine, which serves as an attachment site for hydroxylysine-linked glycosylation. This glycosylation process is initiated by collagen galactosyltransferases from the GT25 family, also known as GLT25D or COLGALT proteins. Despite their biological importance, efficient methods for expressing and isolating GLT25Ds have yet to be fully developed, and the biochemical mechanisms underlying their function still need to be better understood. To address this, we performed sequence alignment and phylogenetic analyses of GLT25Ds across vertebrates, invertebrates, and viruses. Using sponge (<em>amphimedon queenslandica</em>) GLT25D as a model, we established a bacterial expression, purification, and assay protocol. Sponge GLT25D expressed robustly in <em>E. coli</em> strain BL21 and demonstrated enzymatic activity comparable to human GLT25D1 from mammalian cells. Kinetic parameters and the effects of time, temperature and pH on enzymatic activity were characterized for both enzymes. AlphaFold structural modeling and sequence alignment revealed an EXD motif and a conserved leucine in a pocket of the second Rossmann-fold domain of sponge GLT25D, suggesting this pocket as the active site. Using the standardized bacterial expression, purification, and assay protocol, we screened GLT25Ds from various vertebrate and invertebrate species. Notably, the sea walnut (<em>mnemiopsis leidyi</em>) GLT25D exhibited superior expression levels and robust enzymatic activity. This established method provides a strong foundation for future bioengineering efforts, structure-function analyses, and the development of GLT25D inhibitors.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106685"},"PeriodicalIF":1.4,"publicationDate":"2025-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143190255","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

Differentially labeled flaviviral protease-cofactor complex for NMR spectroscopic applications

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-02-02 DOI: 10.1016/j.pep.2025.106684

Ajith Kumar

{"title":"Differentially labeled flaviviral protease-cofactor complex for NMR spectroscopic applications","authors":"Ajith Kumar","doi":"10.1016/j.pep.2025.106684","DOIUrl":"10.1016/j.pep.2025.106684","url":null,"abstract":"<div><div>Flaviviruses such as Dengue, Zika and West-Nile viruses have a positive strand RNA genome which is translated to a polyprotein inside the host cell. The viral polypeptide is matured to its constituents by the enzymatic action of NS2B-NS3 protease-cofactor complex. The flaviviral protease-cofactor complex attracted a lot of interest recently because of its potential for therapeutic intervention and the unique nature of catalysis where the peptide cofactor regulates the enzymatic activity. Obtaining the enzyme and cofactor differentially labeled with naturally abundant nuclei and NMR active nuclei respectively will be helpful in reducing the spectral complexity by making the enzyme invisible in a multidimensional NMR spectrum while only showing peaks from the cofactor. This will enable one to study the properties of the cofactor in isolation using NMR spectroscopy. Here, I have used a strategy for selectively labeling the cofactor within the complex with NMR active nuclei while peaks from the enzyme were rendered invisible. The protocol used here takes advantage of an ‘on-column unfolding’ step during the initial Ni-NTA chromatography to separate the enzyme and cofactor in unfolded conditions. The labeled cofactor was then allowed to fold in the presence of an unlabeled enzyme to obtain a differently labeled complex. We compared the <sup>1</sup>H-<sup>15</sup>N HSQC spectrum of the differently labeled, wild type and free cofactor to ensure that the cofactor attained the desired fold within the complex. The protocol is scalable, inexpensive and can be applied to other two-component enzyme systems where a peptide cofactor is essential for the folding of an enzyme.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106684"},"PeriodicalIF":1.4,"publicationDate":"2025-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143103838","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 and characterization of the intrinsically disordered Arabidopsis thaliana protein SOG1

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-31 DOI: 10.1016/j.pep.2025.106678

Kim Mignon , Margot Galle , Rani Van der Eecken , Sarah Haesaerts , Manon Demulder , Henri De Greve , Lieven De Veylder , Remy Loris

{"title":"Purification and characterization of the intrinsically disordered Arabidopsis thaliana protein SOG1","authors":"Kim Mignon , Margot Galle , Rani Van der Eecken , Sarah Haesaerts , Manon Demulder , Henri De Greve , Lieven De Veylder , Remy Loris","doi":"10.1016/j.pep.2025.106678","DOIUrl":"10.1016/j.pep.2025.106678","url":null,"abstract":"<div><div>SOG1, a transcription factor consisting of a folded NAC (NAM-ATAF-CUC2) domain and an intrinsically disordered C-terminal domain (CTD), co-ordinates the DNA damage response in plants. Here we compare different methods to express and purify recombinant full length <em>Arabidopsis thaliana</em> SOG1. Expression in Sf9 insect cells results in a protein that contains a phosphorylated site that is possibly located on the T423 site in the CTD. This site is reported to be phosphorylated <em>in planta</em> upon aluminium toxicity stress and may affect the transcriptional activity of SOG1 in an yet undetermined way. Expression of SOG1 in <em>E. coli</em> BL21 (DE3) leads to the formation of inclusion bodies, a problem that is resolved by using a cleavable SUMO solubility tag. The resulting protein is not phosphorylated and represents the transcriptional inactive state of SOG1. Both protein preparations show similar CD spectra and melting temperatures. SEC-MALS determined that the proteins, like other NAC transcription factors, form a dimer in solution. Both proteins are also highly non-globular as determined by analytical SEC and are likely stretched out due to their disordered CTD. In electromobility shift assays, both insect and <em>E. coli</em> purified SOG1 proteins bind to a DNA fragment from the promoter region of <em>SMR5</em>, a well established target gene of SOG1, showing the functionality of both purified proteins.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106678"},"PeriodicalIF":1.4,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143075110","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

Purification and characterization of full-length monomeric TEC family kinase, ITK

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-31 DOI: 10.1016/j.pep.2025.106682

Udumbara M. Rathnayake , Junya Wada , Vanessa E. Wall , Jane Jones , Lisa M. Jenkins , Amy H. Andreotti , Lawrence E. Samelson

{"title":"Purification and characterization of full-length monomeric TEC family kinase, ITK","authors":"Udumbara M. Rathnayake , Junya Wada , Vanessa E. Wall , Jane Jones , Lisa M. Jenkins , Amy H. Andreotti , Lawrence E. Samelson","doi":"10.1016/j.pep.2025.106682","DOIUrl":"10.1016/j.pep.2025.106682","url":null,"abstract":"<div><div>An early step in the activation of T cells via the T cell antigen receptor is the phosphorylation and activation of phospholipase C-γ1 (PLC-γ1) by the TEC family tyrosine kinase, interleukin-2 (IL-2) inducible T cell kinase (ITK). PLC-γ1 activation occurs within a multi-protein complex comprised of the enzymes ITK, PLC-γ1, and VAV, and the adapter molecules, LAT, Gads, SLP-76, and NCK. Studies of ITK activation and the role of this heptameric complex in regulating ITK activation and function have not been possible due to the lack of success in the expression and purification of full-length, monomeric ITK protein. In this study, we have produced soluble full-length wild-type ITK protein by co-expressing an N-terminal solubility-tagged ITK construct with a kinase-specific co-chaperone CDC37 in an insect cell line. Although the majority of the purified ITK protein is oligomerized, there is a 13-fold increase in the yield of monomeric protein production compared to the last reported purification. Previous studies suggest that the ITK oligomerization is mediated by intermolecular interactions. We created several mutants to disrupt these self-associations. Expression of one of these, the C96E/T110I mutant, produced 20 times more monomer than the wild-type construct. The <em>in vitro</em> characterization of these protein constructs showed that the purified protein is stable and functional. This successful purification and <em>in vitro</em> characterization of full-length monomeric ITK protein will aid in understanding the mechanism by which ITK is recruited into the heptameric complex and is enabled to phosphorylate and activate PLC-γ1.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106682"},"PeriodicalIF":1.4,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143080758","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

A FRET-based competitive binding assay using coumestrol and the ligand-binding domain of human estrogen receptor alpha tagged with mTurquoise2 efficiently expressed in E. coli with ethanol

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-30 DOI: 10.1016/j.pep.2025.106667

Edith O. López-Romero , Emma L. Arévalo-Salina , César Arcos-Hernández , Yoloxochitl Sánchez-Guevara , Carmen Beltrán , Gloria Saab-Rincón , Takuya Nishigaki

{"title":"A FRET-based competitive binding assay using coumestrol and the ligand-binding domain of human estrogen receptor alpha tagged with mTurquoise2 efficiently expressed in E. coli with ethanol","authors":"Edith O. López-Romero , Emma L. Arévalo-Salina , César Arcos-Hernández , Yoloxochitl Sánchez-Guevara , Carmen Beltrán , Gloria Saab-Rincón , Takuya Nishigaki","doi":"10.1016/j.pep.2025.106667","DOIUrl":"10.1016/j.pep.2025.106667","url":null,"abstract":"<div><div>The estrogen receptor (ER) is a nuclear receptor and one of the most extensively researched targets in the study of endocrine-disrupting chemicals (EDCs). Many biosensors and bioassays for estrogenic EDCs use the ligand-binding domain of human ERα (LBD-hERα) as a biological recognition element. However, the LBD-hERα is poorly stable and difficult to produce as a functional LBD-hERα in the <em>E. coli</em> expression system. In this study, we efficiently expressed the functional LBD-hERα tagged with the cyan fluorescent protein, mTurquoise2 (LBD-hERα-mTq2) by the addition of ethanol (3 %) to <em>E. coli</em> suspension during protein expression (> 40 times more compared to without ethanol). We found that ethanol not only promoted the proper folding of LBD-hERα-mTq2, but also prevented the proteolysis of poorly folded recombinant proteins. We established a FRET-based binding assay between a fluorescent estrogen, coumestrol, and the LBD-hERα-mTq2, in which the formation of the complex exhibits a significant degree of FRET. A subsequent competitive binding assay with diethylstilbestrol demonstrates that our system successfully functions as a simple and reliable bioassay to detect estrogenic EDCs.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106667"},"PeriodicalIF":1.4,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143075100","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

Preparation and characterization of LGR5 LOOP region-specific nanobodies

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-30 DOI: 10.1016/j.pep.2025.106680

Li Jia , Huarui Qiao , Yuting Ding , Qianqian Cui , Yingjun Wang , Jing Geng , Junming Tang , Jianfeng Xu , Yuanyuan Dai , Yong Geng

{"title":"Preparation and characterization of LGR5 LOOP region-specific nanobodies","authors":"Li Jia , Huarui Qiao , Yuting Ding , Qianqian Cui , Yingjun Wang , Jing Geng , Junming Tang , Jianfeng Xu , Yuanyuan Dai , Yong Geng","doi":"10.1016/j.pep.2025.106680","DOIUrl":"10.1016/j.pep.2025.106680","url":null,"abstract":"<div><div>Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), also known as G-protein-coupled receptor 49 (GPR49), is a class A G-protein-coupled receptor (GPCR) that plays a pivotal role in embryonic development and functions as a marker for adult stem cells in various tissues and organs. LGR5 possesses a large extracellular domain (ecto-domain) enriched with leucine-rich repeats (LRR), primarily responsible for binding to ligands such as R-spondins. The C-terminal LRR extracellular LOOP region of LGR5 refers to the loop structure connecting the C-terminus of LGR5 to the first transmembrane helix. As the LOOP region is located extracellularly, it is readily accessible to exogenous molecules such as antibodies, nanobodies, or small-molecule drugs. In this study, we successfully expressed and purified the LGR5 LOOP region protein in a prokaryotic expression system. The purified protein was subsequently used as an antigen to immunize camels, leading to the generation of nanobodies. These nanobodies are composed solely of the variable domain of the heavy-chain antibody (VHH), with a molecular weight of approximately 15 kDa. Using the purified LGR5 LOOP region protein as an antigen, we isolated nanobodies that specifically bind to it. Subsequent assays demonstrated that the selected nanobody, NB 4C4 and NB 3E8, specifically targeted the LGR5 LOOP region, exhibited an inhibitory effect on β-catenin-mediated Wnt signaling to a certain extent. This study provides insights for the development of LGR5-targeted diagnostic reagents and antibody-based therapeutic strategies.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106680"},"PeriodicalIF":1.4,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143075103","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

Screening and preparation of nanobodies for SIGLEC-15 detection

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-27 DOI: 10.1016/j.pep.2025.106679

Shuaiying Zhao , Lingyun Li , Zhongyun Lan , Xiangyun Hou , Ruimin Huang , Quanfang Jin , Qiting Huang , Li Jia , Yingying Kong , Jianchuan Wen , Huarui Qiao , Yiang Wang , Yiwen Xu , Dongna Zhang , Yong Geng , Jianfeng Xu , Yuanyuan Dai

{"title":"Screening and preparation of nanobodies for SIGLEC-15 detection","authors":"Shuaiying Zhao , Lingyun Li , Zhongyun Lan , Xiangyun Hou , Ruimin Huang , Quanfang Jin , Qiting Huang , Li Jia , Yingying Kong , Jianchuan Wen , Huarui Qiao , Yiang Wang , Yiwen Xu , Dongna Zhang , Yong Geng , Jianfeng Xu , Yuanyuan Dai","doi":"10.1016/j.pep.2025.106679","DOIUrl":"10.1016/j.pep.2025.106679","url":null,"abstract":"<div><div>As an important member of the Siglec family, SIGLEC-15 plays an important role in osteoclast differentiation, bone remodeling, and tumor immune evasion. In the tumor microenvironment, SIGLEC-15 functions independently of the B7-H1/PD-1 pathway. In this study, the SIGLEC-15 fusion protein (SIGLEC-15-Fc) was successfully expressed and purified using a eukaryotic expression system. This protein was then used as an antigen to immunize camelids, inducing the production of specific nanobodies (VHHs) targeting SIGLEC-15. The resulting nanobodies exhibited a molecular weight of approximately 15 kDa. After screening, we identified two nanobody strains, Nb1C8 and Nb2D7, both of which bind SIGLEC-15 without competition. We confirmed the nanobodies’ high affinity and stability through Octec platform and stability analyses. Flow cytometry analysis demonstrated that Nb1C8 and Nb2D7 specifically binds to SIGLEC-15 which naturally expressed on bladder cancer cells. This study marks the first development of nanobodies specifically targeting SIGLEC-15, providing a solid foundation for the development of SIGLEC-15-related diagnostic tools and antibody therapeutics.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106679"},"PeriodicalIF":1.4,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143067526","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

Praesto Jetted A50 HipH, a mild pH elution protein A resin, exhibits improved aggregate separation capability and protein elution from it shows unique response to mobile phase additive sodium chloride

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-27 DOI: 10.1016/j.pep.2025.106677

Wenwen Lu, Yan Wan, Yifeng Li

{"title":"Praesto Jetted A50 HipH, a mild pH elution protein A resin, exhibits improved aggregate separation capability and protein elution from it shows unique response to mobile phase additive sodium chloride","authors":"Wenwen Lu, Yan Wan, Yifeng Li","doi":"10.1016/j.pep.2025.106677","DOIUrl":"10.1016/j.pep.2025.106677","url":null,"abstract":"<div><div>Protein A affinity chromatography has been widely used for product capture in monoclonal antibody (mAb), bispecific antibody (bsAb) and Fc-fusion protein purification. However, the low pH (i.e., 3.0–3.5) required for elution may cause aggregation and/or truncation for pH-sensitive molecules. Praesto Jetted A50 HipH from Purolite is a newly launched Protein A resin whose ligand is engineered to enable antibody/Fc-fusion elution at a milder pH (i.e., 4.6 or above) and therefore is more suitable to pH-sensitive molecules. In the current study, we demonstrated that this new Protein A resin, besides allowing mild elution, also possesses improved aggregate separation capability in comparison to traditional Protein A resins. While traditional Protein A resins generally lack any aggregate separation capability, Jetted A50 HipH can remove up to 70% of the aggregates in the load while maintaining good monomer recovery. In addition, we discovered that protein elution from Jetted A50 HipH column responded to mobile phase additive sodium chloride differently from that from traditional Protein A columns (elution was promoted rather than suppressed). This property enables elution at further increased pH (i.e., 5) when proper amount of sodium chloride is included in the elution buffer. Thus, Jetted A50 HipH is a better choice than traditional Protein A resins for pH-sensitive and/or aggregation-prone mAbs, bsAbs and Fc-fusion proteins.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106677"},"PeriodicalIF":1.4,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143047668","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