Protein expression and purification最新文献

Efficient synchronization of high expression and site-specific biotinylation of the PTBP1 RRM3/4 domain PTBP1 RRM3/4结构域的高表达和位点特异性生物素化的高效同步

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-06-05 DOI: 10.1016/j.pep.2025.106755

Zhangheng Ye , Hong Gu , Yuanhang Wang, Yingyi Liu, Yilan Huang, Meiyi Mao, Lan Sun, Mingxing Huang

{"title":"Efficient synchronization of high expression and site-specific biotinylation of the PTBP1 RRM3/4 domain","authors":"Zhangheng Ye , Hong Gu , Yuanhang Wang, Yingyi Liu, Yilan Huang, Meiyi Mao, Lan Sun, Mingxing Huang","doi":"10.1016/j.pep.2025.106755","DOIUrl":"10.1016/j.pep.2025.106755","url":null,"abstract":"<div><div>Polypyrimidine tract-binding protein 1 (PTBP1), a key regulator of mRNA alternative splicing, has emerged as a promising therapeutic target for neurodegenerative diseases and cancer treatment. Given the critical role of its RRM4 domain's lysine residues in binding to polypyrimidine single-stranded RNA, nonspecific biotinylation could potentially alter PTBP1's activity. Here, we describe a method for the concurrent high-level expression and site-specific biotinylation of the PTBP1 RRM3/4 domain using the pQE80L vector and AVB101 host strain. By optimizing codon usage and GC content, we achieved a maximum expression level of approximately 85 μg/mL in bacterial culture, with over 99 % purity and a 72.4 % biotinylation rate. The recombinant 6His-Avi<sup>Biotin</sup>-PTBP1 demonstrated the ability to emit chemiluminescence when coupled with HRP-streptavidin, to be immunoprecipitated by streptavidin magnetic beads, and to interact specifically with (CU)<sub>8</sub> RNA, exhibiting a dissociation constant (K<sub>D</sub>) of 38 nM as determined by Bio-Layer Interferometry (BLI). Collectively, these results indicate that the recombinant 6His-Avi<sup>Biotin</sup>-PTBP1 is suitable for inhibitor screening and kinetic parameter analysis.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"234 ","pages":"Article 106755"},"PeriodicalIF":1.4,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144239799","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Efficient production of functional proaerolysin in E. coli. 大肠杆菌中功能性原溶素的高效生产。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-06-04 DOI: 10.1016/j.pep.2025.106754

Quynh Thi-Huong Pham, Ayako Tagawa, Narumi Iwata, Masaru Nakao, Yusuke Miyanari

{"title":"Efficient production of functional proaerolysin in E. coli.","authors":"Quynh Thi-Huong Pham, Ayako Tagawa, Narumi Iwata, Masaru Nakao, Yusuke Miyanari","doi":"10.1016/j.pep.2025.106754","DOIUrl":"https://doi.org/10.1016/j.pep.2025.106754","url":null,"abstract":"<p><p>Proaerolysin is a bacterial toxin produced by Aeromonas hydrophila that specifically binds to GPI-anchored proteins on the plasma membrane, forming transmembrane pores that induce cell death. Leveraging this unique property, proaerolysin is widely used in diagnostic tests for paroxysmal nocturnal hemoglobinuria (PNH), a disease caused by somatic mutations in the PIGA, a gene involved in the biosynthesis of GPI anchors. Beyond diagnostics, proaerolysin has recently been applied in basic research, including its use as a counter-selection agent in genetic manipulations and as an engineered nanopore for single-molecule detection. Although the bacterial expression and purification of proaerolysin have been previously reported, the yields were low due to its low solubility. Here, we demonstrate that using the SHuffle E. coli strain, which facilitates the disulfide bond formation in the cytoplasm, significantly improves the solubility and proper folding of proaerolysin. We achieved a high yield of proaerolysin, approximately 3 mg from a 50 mL bacterial culture, with a purity exceeding 99%. The functionality of recombinant proaerolysin was confirmed through testing in mouse embryonic stem cells (mESCs), demonstrating that this high-yield production method provides a reliable and cost-effective source of functional proaerolysin for a wide range of biotechnological applications.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106754"},"PeriodicalIF":1.4,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144249359","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

Strategies for generating soluble and monomeric samples of Ycf1p NBD2 生成Ycf1p NBD2可溶性和单体样品的策略

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-06-03 DOI: 10.1016/j.pep.2025.106752

Sarah E.S. Quail , Jeffrey Youn , Voula Kanelis

{"title":"Strategies for generating soluble and monomeric samples of Ycf1p NBD2","authors":"Sarah E.S. Quail , Jeffrey Youn , Voula Kanelis","doi":"10.1016/j.pep.2025.106752","DOIUrl":"10.1016/j.pep.2025.106752","url":null,"abstract":"<div><div>The yeast cadmium factor 1 protein (Ycf1p) is an ATP-binding cassette (ABC) transporter that is located in the vacuolar membrane and is responsible for transporting glutathione-conjugated metals from the cytoplasm into the vacuole. Ycf1p contains the ABC core structure of two transmembrane domains (TMD1, TMD2) and two nucleotide-binding domains (NBD1, NBD2). As a member of the C-subfamily of ABC proteins (ABCC), Ycf1p also contains an N-terminal extension comprised of an additional TMD (TMD0) and L0 linker. Although high-resolution structures of many ABC transporters have been determined, the NBDs can be at low resolution in cryo-EM maps. Thus, studies of the isolated cytosolic NBDs are crucial for obtaining molecular-level details of the dynamics of these catalytic entities, for example. In this study, we present a scheme for obtaining samples of NBD2 from the yeast cadmium factor 1 protein (Ycf1p) in a soluble, monomeric, and functional form. While production of NBD1 from Ycf1p and other ABC proteins has been accomplished, generating samples of NBD2 from different ABC proteins has been elusive for the most part, particularly for ABCC proteins. We show that NBD2 preparation necessitates minimizing dimerization and aggregation of the protein at multiple steps during the purification, which is accomplished by employing a solubility tag, eliminating nucleotides from the buffers, and limiting the duration of spin concentrating steps. This work lays the foundation for detailed studies of Ycf1p NBD2 and provides an outline for optimizing the generation of samples of NBD2 from other ABC proteins.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"234 ","pages":"Article 106752"},"PeriodicalIF":1.4,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144231596","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Expression, purification and functional study of mycobacteriophage D29 histidine-asparagine-histidine endonuclease 分支噬菌体D29组氨酸-天冬酰胺-组氨酸内切酶的表达、纯化及功能研究。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-06-02 DOI: 10.1016/j.pep.2025.106753

Bo Fu, Lvming Wu, Xin Wang, Hongbin Sun

引用次数: 0

Bioinformatics analysis and molecular cloning of squalene synthase from Simaroubaceae 角鲨烯合成酶的生物信息学分析及分子克隆。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-05-30 DOI: 10.1016/j.pep.2025.106751

Yiqing Zhu , Shiyu Wu , Yi Zhang , Shiyang Zhang , Qianyu Zhou , Weidong Zhang , Jinxin Wang , Zhimin Hu

{"title":"Bioinformatics analysis and molecular cloning of squalene synthase from Simaroubaceae","authors":"Yiqing Zhu , Shiyu Wu , Yi Zhang , Shiyang Zhang , Qianyu Zhou , Weidong Zhang , Jinxin Wang , Zhimin Hu","doi":"10.1016/j.pep.2025.106751","DOIUrl":"10.1016/j.pep.2025.106751","url":null,"abstract":"<div><div>Quassinoids are a class of highly oxygenated triterpenoids with C-18, C-19, C-20, C-22, or C-25 skeletons. Squalene synthase (SQS), a key enzyme in the quassinoids biosynthetic pathway, serves as the first step in controlling carbon flux flow into downstream quassinoids. In this study, we screened and analyzed the SQSs from the transcriptome data of the Simaroubaceae family, including AaSQS, BjSQS, AeSQS, QaSQS, ElSQS, and SaSQS. Bioinformatics analysis showed that these SQSs contain C-terminal transmembrane regions and exhibit over 80 % sequence identity with that of <em>Glycyrrhiza glabra</em> (GgSQS1). Phylogenetic analysis revealed that the SQSs from the Simaroubaceae family are more closely related to <em>Malus domestica</em> and <em>Crataegus pinnatifida</em> within the Rosales clade, suggesting an evolutionary trajectory that corroborates the taxonomic classification of Simaroubaceae within the Rosanae superorder. To characterize the function of the SQS from the Simaroubaceae family, the full-length <em>AaSQS</em> was cloned. A soluble AaSQS was obtained by expressing the truncated version lacking 24 amino acids at the C-terminal region in <em>Escherichia coli</em>. Functional analysis showed that AaSQS could catalyze the production of squalene from farnesyl pyrophosphate (FPP). Structural prediction and molecular docking indicated that residues S50, F51, L205, N209, R212, C286, P289, and M313 may be key catalytic residues in AaSQS. Further mutation analysis revealed that all mutants except C286A showed reduced squalene accumulation compared to the wild type (WT). These results provide guidance for quassinoid biosynthesis through metabolic engineering and synthetic biology strategies, particularly by engineering key amino acid residues to enhance enzymatic activity. This study advances the understanding of SQSs in the Simaroubaceae family and provides an important foundation for the study of quassinoid biosynthesis.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"234 ","pages":"Article 106751"},"PeriodicalIF":1.4,"publicationDate":"2025-05-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144199861","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Expression and purification of functional recombinant Cullin1-Rbx1/2 in E. coli 功能性重组Cullin1-Rbx1/2在大肠杆菌中的表达与纯化

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-05-28 DOI: 10.1016/j.pep.2025.106750

Fangyu Zhao , Chuntong Li , Xu Li , Luyu Shi , Yingyue Zhang , Han Wang , Shuzhe Sun , Lu-Jun Liang

{"title":"Expression and purification of functional recombinant Cullin1-Rbx1/2 in E. coli","authors":"Fangyu Zhao , Chuntong Li , Xu Li , Luyu Shi , Yingyue Zhang , Han Wang , Shuzhe Sun , Lu-Jun Liang","doi":"10.1016/j.pep.2025.106750","DOIUrl":"10.1016/j.pep.2025.106750","url":null,"abstract":"<div><div>Protein ubiquitination is a crucial post-translational modification in eukaryotes that is mediated by E1-E2-E3 enzymatic cascades and regulates a wide range of cellular processes. Cullin-RING E3 ligases (CRLs) represent the largest family of RING-type E3 ligases and play pivotal roles in these processes. Generating full-length, active Cullin1-Rbx1 (CRL1) is essential for biochemical and biophysical investigations. In this study, we developed an efficient strategy to produce functional CRL1 complexes from <em>E. coli</em>, by fusing an MsyB protein to N-terminus of Cullin1 to improve its solubility. The recombinant CRL1 demonstrated full functionality, successfully assembling with substrate receptor Skp1-Skp2-Cks1 and mediating the polyubiquitination of Ub-p27-degron substrate. Using recombinant CRL1, we found that phosphorylation at Ser65 inhibited the CRL1-UBE2R1 mediated ubiquitin chain elongation on Ub-p27-degron. Furthermore, using the same strategy, we successfully generated Cullin1-Rbx1 mutants and Cullin1-Rbx2 complexes, thereby expanding the applicability of our method. Collectively, this work establishes a rapid and cost-effective platform for the production of CRL1 complexes, facilitating structural and functional studies.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"233 ","pages":"Article 106750"},"PeriodicalIF":1.4,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144177701","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 simple protocol for controlling protein deuteration for neutron scattering 控制中子散射中蛋白质氘化的简单规程

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-05-28 DOI: 10.1016/j.pep.2025.106749

Satoru Fujiwara , Motoyasu Adachi , Yasunobu Sugimoto

{"title":"A simple protocol for controlling protein deuteration for neutron scattering","authors":"Satoru Fujiwara , Motoyasu Adachi , Yasunobu Sugimoto","doi":"10.1016/j.pep.2025.106749","DOIUrl":"10.1016/j.pep.2025.106749","url":null,"abstract":"<div><div>Protein deuteration is a crucial technique in biological studies using neutron scattering/diffraction. In neutron crystallography, the use of deuterated proteins not only reduces the background due to hydrogen atoms but also prevents the cancellation of the scattering length density due to the negative density of hydrogen atoms. In small-angle neutron scattering, it allows the structures of individual components of a protein complex to be studied. Furthermore, in quasielastic neutron scattering, it allows one to measure the dynamics of specific components (or regions) in protein complexes (or a protein). Protocols for protein deuteration employ the expression system in <em>E. coli</em> cultured either in minimal media containing, for example, deuterated glycerol or in rich media containing deuterated algal hydrolysate (algal peptone). While the protocols using the minimal media are the mainstream, the protocols using the rich media allow easy control of deuteration. Here, we optimize the procedure for preparing algal peptone, and describe a protocol for protein deuteration using algal peptone. It is shown that ∼96 % deuteration of the test protein, α-synuclein (αSyn), can be routinely achieved using this protocol. The similarity of the structures of deuterated and hydrogenated αSyn was verified by the small-angle X-ray scattering measurements. Furthermore, the degrees of deuteration can be controlled simply by mixing the hydrogenated and deuterated peptone in the media, and hydrogen labeling of specific amino-acid residues is possible by adding hydrogenated amino acids to the media. The protocol described here is thus useful as a complementary method to those using the minimal media.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"233 ","pages":"Article 106749"},"PeriodicalIF":1.4,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144177702","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 of CYP27A1 recombinant protein expression CYP27A1重组蛋白表达的优化。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-05-26 DOI: 10.1016/j.pep.2025.106748

Johanna E. Papa, Lindsay R. Vaughn, Jackson L. Bartholomew-Schoch, Emma K. Stone, Megen A. Culpepper, Michael J. Reddish

{"title":"Optimization of CYP27A1 recombinant protein expression","authors":"Johanna E. Papa, Lindsay R. Vaughn, Jackson L. Bartholomew-Schoch, Emma K. Stone, Megen A. Culpepper, Michael J. Reddish","doi":"10.1016/j.pep.2025.106748","DOIUrl":"10.1016/j.pep.2025.106748","url":null,"abstract":"<div><div>Human mitochondrial cytochrome P450 27A1 is a monooxygenase enzyme that oxidizes bile acids and other sterol derivatives. The enzyme plays an important role in sterol metabolism and is a potential target for clinical therapies related to metabolic conditions and certain cancers. To support the development of such therapies, detailed structural and functional studies of the enzyme should be pursued. Producing large quantities of purified, recombinant enzyme would enable these studies. Recombinant production of human cytochrome P450 27A1 in <em>E. coli</em> is challenging due to the enzyme being membrane associated. This work explores the optimization of human cytochrome P450 27A1 expression in <em>E. coli</em> by systematically testing the effects of cell strain, expression temperature, concentrations of induction reagents, and expression times. Western blot analysis is used to investigate the effects of variable changes prior to purification. <em>E. coli</em> cell strain (switching to C41(DE3)) appears to have the largest positive effect on overall yield. Increasing δ-aminolevulinic acid concentration (induces heme synthesis) also leads to significantly increased yields. Decreasing expression time decreases the amount of higher order cytochrome P450 aggregates that are formed. The combination of these changes is a more robust expression protocol with three major advantages: decreased expression time, lower aggregate to monomer ratios, and increased overall yield.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"233 ","pages":"Article 106748"},"PeriodicalIF":1.4,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144174687","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 magnetic particle-based chemiluminescence immunoassay for quantitative determination of hepatitis C virus core antigen 磁颗粒化学发光免疫法定量测定丙型肝炎病毒核心抗原的建立

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-05-22 DOI: 10.1016/j.pep.2025.106747

Ye Xu , Yating Liu , Jianrong Zhu , Chufan Tan , Ting Wan , Songhui Zhou , Mengrong Cheng , Jiao Zheng , Rushi Liu

{"title":"Development of magnetic particle-based chemiluminescence immunoassay for quantitative determination of hepatitis C virus core antigen","authors":"Ye Xu , Yating Liu , Jianrong Zhu , Chufan Tan , Ting Wan , Songhui Zhou , Mengrong Cheng , Jiao Zheng , Rushi Liu","doi":"10.1016/j.pep.2025.106747","DOIUrl":"10.1016/j.pep.2025.106747","url":null,"abstract":"<div><h3>Objectives</h3><div>Development of an immunoassay for quantitation detecting the highly conserved hepatitis C core antigen (HCVcAg).</div></div><div><h3>Methods</h3><div>Anti-HCVcAg monoclonal antibodies (mAbs) were developed and evaluated using enzyme-linked immunosorbent assay (ELISA), and the chemiluminescence enzyme immunoassay (CLEIA) was also proposed. The serum HCVcAg was measured to evaluate and analyze the performance of CLEIA and analysis of the mAb pair was evaluated by sequencing of the antibody variable region.</div></div><div><h3>Results</h3><div>The prokaryotically expressed HCVcAg was purifed and three HCVcAg mAbs against HCVcAg with excellent detection performance were obtained after immunizing BALB/c mice. MAb 4D11 and 28B10 were selected as respective capture and detection antibodies for HCVcAg measurement by CLEIA (detection range, 0.1–256 μg/L). The results for CLEIA and fluorescent PCR used in hospitals demonstrated excellent consistency (<em>K</em> = 0.801, <em>P</em> < 0.01). The variable region genes of the heavy chain (VH) and light chain (VL) for the mAb pair were successfully sequenced.</div></div><div><h3>Conclusion</h3><div>The developed mAbs and CLEIA are expected to become a rapid and economical clinical diagnostic tool for HCVcAg detection.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"233 ","pages":"Article 106747"},"PeriodicalIF":1.4,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144138685","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

High-yield production of recombinant CLCF1 protein fused with human serum albumin in animal cells and toxicity evaluation in rodents 重组CLCF1蛋白与人血清白蛋白融合在动物细胞中的高产生产及其对啮齿动物的毒性评价。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-05-18 DOI: 10.1016/j.pep.2025.106740

Jeong Min Lee , Jae Sook Kang , Yong Ryoul Yang , Ji Hoon Park

{"title":"High-yield production of recombinant CLCF1 protein fused with human serum albumin in animal cells and toxicity evaluation in rodents","authors":"Jeong Min Lee , Jae Sook Kang , Yong Ryoul Yang , Ji Hoon Park","doi":"10.1016/j.pep.2025.106740","DOIUrl":"10.1016/j.pep.2025.106740","url":null,"abstract":"<div><div>Recombinant protein based biopharmaceutics have been developed as therapeutics of various diseases, especially cancer, diabetes, infectious diseases, and autoimmune diseases. In this study, we conducted a study for the development of biopharmaceuticals based on the CLCF1 protein. First, we established strategies for producing recombinant human CLCF1 protein by transient gene expression in ExpiCHO-S™ Cells and Expi293F™ Cells. For the secretion of CLCF1 protein, we established strategies that human CRLF1 or sCNTFR with CLCF1 protein were co-expressed. As a result, the CLCF1 protein formed a complex with CRLF1 or sCNTFR, which was successfully secreted. Furthermore, the productivity of CLCF1 protein was significantly increased. The ratio of co-transfected plasmids, temperature, CO<sub>2</sub> level and time of harvest were explored, so that the productivity of CLCF1 was remarkably increased 7-fold from 3 mg/L to 22 mg/L. Next, we generated recombinant CLCF1 fusion protein with HSA (Albumin CLCF1) considering the improvement of pharmacokinetic properties and the proven production method in GMP facilities. We evaluated the biological activity of various CLCF1 proteins. In consideration of protein productivity, physical properties, and efficacy, we conducted a single intravenous administration of 4 types of proteins in Sprague-Dawley rats to evaluate the short-term toxicity. As a result, no toxicity related CLCF1 proteins was observed based on the behavior sign observation and body weight changes. In conclusion, we successfully established the strategies of production and characterization of biologically active recombinant CLCF1 proteins in mammalian cells as potential biotherapeutics.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"233 ","pages":"Article 106740"},"PeriodicalIF":1.4,"publicationDate":"2025-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111266","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