Yiwen Xu , Yiang Wang , Zhongyun Lan , Yaxin Tuo , Siyu Zhou , Shuaiying Zhao , Yunfeng Liu , Yingying Kong , Huarui Qiao , Jianfeng Xu , Yuanyuan Dai , Yong Geng
{"title":"Screening and functional studies of Norrin-related nanobodies","authors":"Yiwen Xu , Yiang Wang , Zhongyun Lan , Yaxin Tuo , Siyu Zhou , Shuaiying Zhao , Yunfeng Liu , Yingying Kong , Huarui Qiao , Jianfeng Xu , Yuanyuan Dai , Yong Geng","doi":"10.1016/j.pep.2025.106717","DOIUrl":"10.1016/j.pep.2025.106717","url":null,"abstract":"<div><div>Norrin is a crucial regulator of the Wnt/β-catenin signaling pathway, playing a key role in retinal vascular development, blood-brain barrier maintenance, and neuroprotection. In this study, a Norrin fusion protein (Norrin-Fc) was successfully expressed and purified using an insect cell expression system. Camels were immunized with purified Norrin-Fc to generate nanobodies, which consist solely of the heavy chain variable domains of heavy chain antibodies (VHH) with a molecular weight of approximately 15 kDa. Two nanobody strains, Nb 1C4 and Nb 2B10, were identified for their specific binding to Norrin-Fc, and their binding affinities were further characterized. Flow cytometry analysis confirmed that Nb 1C4 and Nb 2B10 specifically bound to the Norrin-FZD4 fusion protein. Luciferase reporter assay results demonstrated that both nanobodies effectively disrupted LGR4-induced Wnt/β-catenin signaling upon Norrin stimulation. This study represents the first successful development of nanobodies targeting Norrin, providing a strong foundation for the advancement of Norrin-related diagnostic tools and antibody therapeutics.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106717"},"PeriodicalIF":1.4,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143821002","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}
{"title":"The tumor suppressor LZTR1: Its expression, purification and characterization","authors":"Yifang Sun, Yuxuan Jiang, Meng Zhang, Lei Sun","doi":"10.1016/j.pep.2025.106716","DOIUrl":"10.1016/j.pep.2025.106716","url":null,"abstract":"<div><div>Leucine-zipper transcription regulator 1 (LZTR1) serves as a tumor suppressor gene that is highly mutated and has been implicated in the pathogenesis of diverse cancers and developmental disorders. The LZTR1 protein is a member of the BTB-Kelch superfamily and functions as an adaptor that enables the recognition and recruitment of RAS proteins, which are then targeted for ubiquitination by the Cullin3-RING ligase E3 (CRL3) complex. Understanding the assembly mechanisms, substrate recognition patterns, and pathological processes associated with RAS ubiquitination mediated by LZTR1-CUL3 is critical. In this study, we report the expression, purification, and characterization of the LZTR1 to investigate its molecular mechanisms. Our findings demonstrate that the BacMam expression system significantly enhances LZTR1 production and simultaneously improves its stability and solubility. Furthermore, the existence of CRL3 contributes to stabilizing and homogenizing LZTR1 through facilitating the formation of the complex. Additionally, we identified MRAS as a substrate that binds tightly to LZTR1, in contrast to RIT1 and HRAS. Successfully expressing and purifying the full-length CRL3<sup>LZTR1</sup>-MRAS complex provide a foundation for future structural and functional investigations and offer a potential approach for exploring other BTB proteins with similar characteristics.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106716"},"PeriodicalIF":1.4,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143824234","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}
Jing Hu , Wenxuan Feng , Jianchuan Wen , Siyu Zhou , Zengchao Sun , Shuaiying Zhao , Shaojue Guo , Hui Wang , Yong Geng
{"title":"Corrigendum to “High-affinity nanobodies targeting IL-12B for the detection of fluorescence resonance energy transfer” [Protein Express. Purificat. 229 (2025) 106681]","authors":"Jing Hu , Wenxuan Feng , Jianchuan Wen , Siyu Zhou , Zengchao Sun , Shuaiying Zhao , Shaojue Guo , Hui Wang , Yong Geng","doi":"10.1016/j.pep.2025.106714","DOIUrl":"10.1016/j.pep.2025.106714","url":null,"abstract":"","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106714"},"PeriodicalIF":1.4,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907853","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}
Keyi Wang , Jing Huang , Mingzhi Li , Tianxiang Han , Ju Yao , Decheng Kong , Bao Liu , Xiaojin Xia , Yunli Guo , Sha Li , Lingbao Kong
{"title":"Bioinformatics analysis and polyclonal antibody preparation of Mesocricetus auratus DHX30","authors":"Keyi Wang , Jing Huang , Mingzhi Li , Tianxiang Han , Ju Yao , Decheng Kong , Bao Liu , Xiaojin Xia , Yunli Guo , Sha Li , Lingbao Kong","doi":"10.1016/j.pep.2025.106715","DOIUrl":"10.1016/j.pep.2025.106715","url":null,"abstract":"<div><div>DHX30, as members of the DExD/H family, plays a crucial regulatory role in RNA metabolism and antiviral innate immune response. To facilitate comprehensive investigation of DHX30's multifunctional involvement in viral life cycles, a highly specific anti-DHX30 antibody is needed. In this paper, we used bioinformatic analyses combined with the Immune Epitope Database (IEDB) to identify immunodominant B-cell epitopes in the <em>Mesocricetus auratus</em> DHX30 protein. A hydrophilic peptide sequence demonstrating strong antigenic potential was selected for recombinant antigen production. The corresponding DHX30 truncated gene fragment was cloned into the pET-28a vector to generate the pET-28a-DHX30-N expression construct. Following transformation into <em>E. coli</em> BL21(DE3) competent cells, recombinant protein expression was induced and subsequently purified via Ni-NTA affinity chromatography. Six-week-old female KM mice were immunized intraperitoneally with the purified antigen. The antibody titer was determined by indirect ELISA, demonstrating high serum titers. Specificity validation through immunoblotting and immunocytochemical analyses confirmed the antibody's exceptional target recognition capability. These antibodies are expected to develop detection methods for further research.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106715"},"PeriodicalIF":1.4,"publicationDate":"2025-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143812165","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}
Dalton Kiefer , Lucas Bierscheid , Oliver Kask , Calvin Heyl , Shiza Rehman , Jacqueline Carmona , Karen S. Anderson , Petra Fromme
{"title":"Diverse approaches to isolate HLA class I molecules from bacterial inclusion bodies, forming heterotrimeric complexes","authors":"Dalton Kiefer , Lucas Bierscheid , Oliver Kask , Calvin Heyl , Shiza Rehman , Jacqueline Carmona , Karen S. Anderson , Petra Fromme","doi":"10.1016/j.pep.2025.106713","DOIUrl":"10.1016/j.pep.2025.106713","url":null,"abstract":"<div><div>Production of recombinant human leukocyte antigen class I (HLA-I) proteins in vitro is fundamental for molecular immunology. However, HLA-I protein refolding has remained inefficient due to challenges in the assembling of the trimolecular complex. Here, we compare various in vitro refolding methods that address the challenges of intrachain disulfide bond formation and assembly of the complex between the light and heavy chains in the presence of the target peptide. We developed methods that uncouple the oxidation of disulfide bond formation of both subunits of HLA-I, followed by renaturation to promote complex formation. CuSO<sub>4</sub>-catalyzed air oxidation enhances correct disulfide bond formation when the protein is solubilized with <em>N</em>-lauryl-sarcosine (sarkosyl); however, careful removal of sarkosyl did not prevent heavy chain aggregation. We modified the classical method of HLA-I refolding by pre-oxidizing the β<sub>2</sub>m light chain before adding the HLA-I heavy chain and peptide. This method yielded successful complex refolding for HLA-A∗02:01/GILGFVFTL at 24.2 % efficiency, and HLA-C∗12:03/KAYNVTQAF at 14.5 % efficiency. Our results suggest that pre-folded β<sub>2</sub>m improves refolding efficiency of HLA-I molecules. This work presents novel approaches to HLA-I refolding that may be applied to other difficult-to-fold protein complexes.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106713"},"PeriodicalIF":1.4,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143743462","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}
Lidija S. Vrhovac , Maria Levkovets , Vladislav Y. Orekhov , Sebastian Westenhoff
{"title":"Refolding of the Deinococcus Radiodurans phytochrome photosensory module and an extended backbone resonance assignment by solution NMR","authors":"Lidija S. Vrhovac , Maria Levkovets , Vladislav Y. Orekhov , Sebastian Westenhoff","doi":"10.1016/j.pep.2025.106699","DOIUrl":"10.1016/j.pep.2025.106699","url":null,"abstract":"<div><div>Solution NMR reveals the structure and dynamics of biomolecules in solution. In particular, the method can detect changes due to perturbation of the molecules, without limiting effects of frozen particles or crystal environments. Phytochromes are photosensors which control the response to red/far-red light in bacteria, fungi and plants, undergo specific structural changes when photoactivated from the Pr to the Pfr state. While structures of phytochromes have been revealed in both states, the structural mechanism of photoconversion remains incompletely understood. Our previous NMR studies of the entire photosensory core module of the <em>D. radiodurans</em> phytochrome have revealed novel structural changes, but the backbone assignment was incomplete. In particular, a lack of the assignment in the protein core hindered more detailed insight in signaling mechanism. Here, we outline an efficient procedure for the refolding of the three-domain, photosensory core fragment of the <em>D. radiodurans</em> phytochrome in its monomeric form. We find that treatment with guanidinium hydrochloride and subsequent dilution effectively refolds the phytochrome, maintaining its functionality. We characterize the refolded protein with solution NMR spectroscopy newly assigning 27 (44) residues in Pr (Pfr), out of which 12 exhibit notable chemical shift perturbation upon photoactivation. The study presents a functional method for purification and refolding of a multidomain protein and opens the door for further structural and dynamic analysis of phytochromes.</div><div><strong>Author summary</strong></div><div>Refolding of proteins is an established method to increase the deuterium-hydrogen exchange of amid bonds in isotopically labeled proteins, which are located deep in the protein core. Yet, the method has to be optimized for each individual protein and in particular for multidomain proteins it is not trivial to find satisfactory experimental conditions. Here we identify a method to refold a <em>D. radiodurans</em> phytochrome construct and characterize the outcome of the procedure using solution NMR and optical spectroscopy. The quick accessibility on whether the refolded phytochrome was functional or not has been obtained from optical spectra, which also made the screening of a number of additives possible. The procedure led to a significant increase in the number of the assigned residues especially in the protein core, close to the photochemically active chromophore, which enables a more detailed investigation of the structure and dynamics throughout the photocycle of the phytochrome.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106699"},"PeriodicalIF":1.4,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143693126","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}
Yankang Wang , Hongmei Zhang , Wenjing Shi , Yongheng Rong , Weian Mao , Linhan Wang , Wenzhu Tang , Yun Kong , Shengjun Wang
{"title":"High soluble expression and characterization of human GalNAc transferase T2 and T11 in Escherichia coli","authors":"Yankang Wang , Hongmei Zhang , Wenjing Shi , Yongheng Rong , Weian Mao , Linhan Wang , Wenzhu Tang , Yun Kong , Shengjun Wang","doi":"10.1016/j.pep.2025.106712","DOIUrl":"10.1016/j.pep.2025.106712","url":null,"abstract":"<div><div>The efficient expression of soluble glycosyltransferases from mammalian sources in <em>Escherichia coli</em> (<em>E. coli</em>) remains a significant challenge, often resulting in misfolding and the formation of inclusion bodies. In this study, we investigated strategies to enhance the solubility and catalytic activity of human GalNAc-T2 and GalNAc-T11, two <em>O</em>-glycosyltransferases involved in <em>O</em>-glycosylation of glycoproteins. We found that fusion with maltose-binding protein (MBP) and cellulase catalytic domain (Cel-CD), which led to majority of the fusion proteins being soluble, could increase the solubility of the recombinant proteins. Enzyme activity assays revealed that the fusion glycosyltransferase exhibited significantly higher catalytic efficiency than non-fused enzymes. In addition, the influence of GalNAc-T11 lectin domain on substrate specificity was also determined. The presence of lectin domain had no influence on the recognition of specific substrate and the specific activity of GalNAc-T11. This work offers an efficient approach for the large-scale production of human glycosyltransferases with enhanced bioactivity, highlighting its potential for glycosylation engineering of glycoprotein drugs.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106712"},"PeriodicalIF":1.4,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143684805","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}
{"title":"Impact of arginine addition on protein concentration via ultrafiltration","authors":"Teruo Akuta , Yasunori Kurosawa , Yui Tomioka , Tsutomu Arakawa","doi":"10.1016/j.pep.2025.106701","DOIUrl":"10.1016/j.pep.2025.106701","url":null,"abstract":"<div><div>In this study, we examined the effects of arginine (L-ArgHCl) on ultrafiltration performance, a process with practical significance for not only research but also pharmaceutical applications. Specifically, we assessed the yield and filtration rate of ultrafiltration using rabbit and goat polyclonal antibodies (neutral to basic isoelectric points) as well as model proteins, BSA (acidic) and lysozyme (basic). When a 1 mg/mL protein solution was concentrated approximately 10-fold using a standard commercially available centrifugal ultrafiltration device, the addition of L-ArgHCl significantly improved yield at near-neutral buffer pH. The observed order of improvement was: 20 mM L-ArgHCl >100 mM L-ArgHCl ≈0.5 M NaCl > no addition. A similar trend was observed with BSA, whereas lysozyme achieved slightly higher yields at 100 mM L-ArgHCl. In a 40-fold concentration of rabbit polyclonal antibody from 1 mg/mL, 20 mM L-ArgHCl enhanced yield at pH 6 and 7, but had minimal or no effect at pH 7.5. Notably, at pH 8, high yields were achieved without arginine. L-ArgHCl also accelerated the concentration rate at all pH levels, with greater enhancements observed at higher arginine concentrations. These findings suggest that L-ArgHCl mitigates protein precipitation and solubility loss by reducing protein-protein interactions and nonspecific binding to the ultrafiltration membrane. At pH 8, the increased surface charge of the antibody reduced hydrophobicity, further improving solubility. In summary, the addition of L-ArgHCl, particularly near pH 7, effectively enhanced ultrafiltration performance. This provides a practical strategy for improving protein concentration processes.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106701"},"PeriodicalIF":1.4,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143670737","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}
Tianyi Gao , Yun Wang , Tong Zhang , Rou Li , Yue Sun , Kui Zhang , Min Xu , Fei Liu , Boxing Cheng
{"title":"Molecular cloning and functional analysis of a destabilase from Hirudinaria manillensis","authors":"Tianyi Gao , Yun Wang , Tong Zhang , Rou Li , Yue Sun , Kui Zhang , Min Xu , Fei Liu , Boxing Cheng","doi":"10.1016/j.pep.2025.106703","DOIUrl":"10.1016/j.pep.2025.106703","url":null,"abstract":"<div><div>Destabilases are i-type lysozymes with isopeptidase activity and antibacterial and thrombolytic functions. In recent years, destabliases have been identified in an increasing number of invertebrates. <em>Hirudinaria manillensis</em> belonging to the Annelida, as one of the origins of leeches used in traditional Chinese medicine, which has high medicinal value, there have been few reports on the <em>H. manillensis</em> destabliase. In this study, the cDNA sequence of <em>Hmdestabilase</em> was cloned from the salivary glands of <em>H. manillensis</em>. The 3D Structural analysis indicated that Hmdestabilase is similar to other i-type lysozymes in that it adopts an ellipsoidal shape and has a large cleft containing the lysozyme active site. The docking results of Hmdestabilase protein with N-acetylglucosamine trimer molecule have shown that the location and number of hydrogen bonds are one of the key factors for the interaction between the protein and its substrate. The Hmdestabilase fusion protein obtained through the prokaryotic expression system has lysozyme and isopeptidase activities. In addition, Changes in sodium ion concentration in the environment affect the lysozyme activity of Hmdestabilase fusion protein. The above bioinformatic analysis and enzymatic function studies have shown that Hmdestabilase belongs to the i-type lysozyme family. qPCR analysis revealed that blood feeding significantly increased the mRNA expression of <em>Hmdestabilase</em> in the salivary glands of <em>H. manillensis</em>,and successfully priming the innate immune system against harmful microorganisms ingested with food. This study is helpful to elucidate the innate immune response of <em>H. manillensis</em> and promote the artificial breeding of <em>H. manillensis</em>.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"232 ","pages":"Article 106703"},"PeriodicalIF":1.4,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143664386","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}
{"title":"Preparation and NMR characterization of Aβ peptides at pathological pH","authors":"Xinyue He , Yalan Ma , Naixia Zhang , Chen Zhou","doi":"10.1016/j.pep.2025.106704","DOIUrl":"10.1016/j.pep.2025.106704","url":null,"abstract":"<div><div>Alzheimer's disease (AD) is a neurodegenerative disorder marked by the progressive deterioration of cognitive function. Its pathological hallmarks include the formation of amyloid plaques, which are primarily due to the abnormal aggregation of Aβ peptides. However, the propensity of Aβ peptides for aggregation makes the <em>in vitro</em> preparation very challenging, often resulting in low yield, instability, and impurities. Here in this study, we developed an <em>in vitro</em> method for preparing monomeric Aβ peptides to achieve stable and high-purity samples. Specifically, three strategies including the uses of high concentration of protein denaturant urea, alkaline buffer (ammonium carbonate buffer), and organic solvents (acetonitrile, hexafluoroisopropanol) were applied to prevent Aβ aggregation during the purification. Through an optimized production process, we successfully obtained stable and highly pure <sup>15</sup>N, <sup>13</sup>C-doubly labeled monomeric Aβ40 and Aβ42 peptides suitable for NMR data collection at the pathological acidic pH. Overall, the preparation method presented here offer a robust approach for <em>in vitro</em> production of monomeric Aβ peptides with satisfying purity and reproducibility. Meanwhile, the NMR characterization results for Aβ40 and Aβ42 at pH 6.5 provide useful information for the further biophysical studies involving these two peptides.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106704"},"PeriodicalIF":1.4,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143664387","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}