Protein expression and purification最新文献

A new combination approach to extracellular production of 5-aminolevulinic acid for purification and application in alleviating cadmium-induced oxidative stress in maize 细胞外合成5-氨基乙酰丙酸的新方法及其在镉诱导玉米氧化应激中的应用

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-05-09 DOI: 10.1016/j.pep.2025.106736

Di Zhang, Jinjing Li, Xiaofeng Chen, Shuncheng Zhang, Baokang Wu, Jun Fan

{"title":"A new combination approach to extracellular production of 5-aminolevulinic acid for purification and application in alleviating cadmium-induced oxidative stress in maize","authors":"Di Zhang, Jinjing Li, Xiaofeng Chen, Shuncheng Zhang, Baokang Wu, Jun Fan","doi":"10.1016/j.pep.2025.106736","DOIUrl":"10.1016/j.pep.2025.106736","url":null,"abstract":"<div><div>5-Aminolevulinic acid (ALA) is widely applied in agriculture, animal husbandry, medicine, and often manufactured in <em>Escherichia coli</em> for overexpressing ALA synthase (ALAS) from α-proteobacteria. For enhancing extracellular ALA production, several approaches have been exploited. Here, we developed and identified a new combination strategy to increase ALA production in <em>E. coli</em>, including selection of the negatively-charged peptide tag as a <em>C</em>-terminal fusion partner for increasing soluble production of the ALAS codon variant from <em>Rhodobacter sphaeroides</em>, mutation of certain residues to increase the ALAS variant activity, optimization of the signal sequences to facilitate ALA secretion, down-regulation of the <em>hemB</em> to inhibit ALA transformation in one plasmid expression system, and supply of 4 mM dithiothreitol to the culture to increase cells tolerant to the oxidative stress. Under the specified cultural conditions, ALA yield was up to 3.2 g/L in flash flasks. Compared with the added cadmium-induced stress, simultaneous supply of purified ALA improved maize seedlings growth, decreased contents of malondialdehyde and hydrogen peroxide, and increased peroxidase activity, contents of chlorophylls and proline.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"232 ","pages":"Article 106736"},"PeriodicalIF":1.4,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143942435","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

Navigating the Frontier: Advances in monoclonal antibody purity control 导航前沿：单克隆抗体纯度控制的进展

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-05-07 DOI: 10.1016/j.pep.2025.106725

Xiaoyue Song , Huairu Tian , Rongxian Jing , Kunmei Liu , Kailin Xu , Le Guo , Guolin Zhang

{"title":"Navigating the Frontier: Advances in monoclonal antibody purity control","authors":"Xiaoyue Song , Huairu Tian , Rongxian Jing , Kunmei Liu , Kailin Xu , Le Guo , Guolin Zhang","doi":"10.1016/j.pep.2025.106725","DOIUrl":"10.1016/j.pep.2025.106725","url":null,"abstract":"<div><div>This review explores the evolving landscape of monoclonal antibody (mAb) purity assessment, highlighting the advancements in separation techniques aimed at improving resolution, speed, and precision of impurity detection, identification, and quantification.Traditional methods such as SDS-PAGE, isoelectric focusing electrophoresis and size-exclusion chromatography serve as a foundational tool, while advanced chromatographic techniques, including diverse modes of high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE), offer improved capabilities for analyzing mAb structural integrity and impurities. These techniques separate different constituents in the samples based on their physicochemical properties such as size, charge, isoelectric point, hydrophobicity, and structure. The review discusses the principles and progressions of both traditional and advanced separation techniques and demonstrates their applications in large-scale mAb production and antibody-drug conjugate (ADC) analysis. Innovations in chromatography, such as superficially porous particles and novel stationary phases, address challenges like band broadening and protein adsorption.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"232 ","pages":"Article 106725"},"PeriodicalIF":1.4,"publicationDate":"2025-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143937480","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

Refolding of the recombinant IgV domain of CD47 from E. coli for NMR studies 重组大肠杆菌CD47 IgV结构域的NMR研究

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-05-05 DOI: 10.1016/j.pep.2025.106735

Shundene Key , Ethan Neeley , Shri Swaminathan , Nataly P. Podolnikova , Tatiana P. Ugarova , Xu Wang

{"title":"Refolding of the recombinant IgV domain of CD47 from E. coli for NMR studies","authors":"Shundene Key , Ethan Neeley , Shri Swaminathan , Nataly P. Podolnikova , Tatiana P. Ugarova , Xu Wang","doi":"10.1016/j.pep.2025.106735","DOIUrl":"10.1016/j.pep.2025.106735","url":null,"abstract":"<div><div>CD47 is a widely expressed integrin-associated transmembrane protein that regulates many macrophage functions, including phagocytosis. Since many cancer cells overexpress CD47 to evade the immune system, targeting CD47 has been proposed as a strategy to enhance macrophage-mediated destruction of cancer cells. Consequently, developing antagonists that block the CD47-SIRPα interaction has drawn attention. However, the exclusive use of eukaryotic cell culture to produce CD47-IgV precludes isotope enrichment of the domain. This prevents the use of solution NMR as a tool for identifying CD47 inhibitors. Here, we describe a two-step refolding protocol for the CD47-IgV domain from inclusion bodies produced in <em>E. coli</em>. The yield of CD47-IgV is ∼30 mg/L of culture. The refolded domain interacts with the anti-CD47 antibody B6H12 with an affinity similar to glycosylated CD47-IgV produced in mammalian cells and binds the IgV domain of SIRPα. The method allowed us to produce <sup>15</sup>N and <sup>13</sup>C enriched CD47-IgV for NMR. The chemical shift assignments of the CD47-IgV domain backbone atoms confirmed that the refolded protein has the same secondary structure as the crystal structure of CD47-IgV produced in mammalian cells. Furthermore, NMR data showed refolded CD47-IgV interacts with SIRPα-IgV similarly to glycosylated CD47-IgV. The application of this method can advance the progress of biochemical investigations of the interaction between CD47-IgV and SIRPα and will be useful for the discovery of antibodies, small molecules, and peptides targeting the CD47/SIRPα axis in vivo.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"232 ","pages":"Article 106735"},"PeriodicalIF":1.4,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143918475","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

Overcoming fluorescence loss in mEOS-based AAA+ unfoldase reporters through covalent linkage 通过共价连锁克服基于meos的AAA+展开酶报告基因的荧光损失

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-04-28 DOI: 10.1016/j.pep.2025.106724

Isabella R. Walter , Baylee A. Smith , Dominic Castanzo , Matthew L. Wohlever

引用次数: 0

Purification, folding, activity analysis and substrate specificity of Pseudomonas diacylglycerol kinase 假单胞菌二酰基甘油激酶的纯化、折叠、活性分析和底物特异性

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-04-27 DOI: 10.1016/j.pep.2025.106723

Yipeng Chen , Bin Huai , Jin Chuan Wu , Ning Zhang , Yong Wang , Qingxin Li

{"title":"Purification, folding, activity analysis and substrate specificity of Pseudomonas diacylglycerol kinase","authors":"Yipeng Chen , Bin Huai , Jin Chuan Wu , Ning Zhang , Yong Wang , Qingxin Li","doi":"10.1016/j.pep.2025.106723","DOIUrl":"10.1016/j.pep.2025.106723","url":null,"abstract":"<div><div>The structural and functional investigation of bacterial membrane proteins is crucial to the development of antibiotics. Diacylglycerol kinase (DAGK) from <em>Escherichia coli</em> (<em>E. coli</em>) has been extensively studied as a model membrane protein. However, the DAGK from <em>Pseudomonas aeruginosa</em> (PAO1-DAGK) with a 44 % sequence identity to <em>E. coli</em>-DAGK is not well characterized. To explore the properties of PAO1-DAGK, it was successfully expressed in <em>E. coli</em> and was purified in Decyl-β-D-maltoside (DM) micelles followed with characterizations. Chemical cross-linking studies revealed that PAO1-DAGK in DM micelles could form dimers and trimers. The kinase activity of PAO1-DAGK was determined to be 24.2 ± 2.2 U/mg protein in a mixed-micelle system. The effects of pH and temperature on the activity of PAO1-DAGK were also investigated, respectively. PAO1-DAGK in DM micelles exhibited good stability at pH 6.0–10.0 and below 45 °C. Substrate specificity measurements indicated that PAO1-DAGK demonstrated a clear preference for medium-chain diacylglycerols (DAGs) in the mixed-micelle system, with sn-1,2-Dihexanoylglycerol (DiC6) being the most favored substrate. Molecular docking results demonstrated the interactions between DAGs and PAO1-DAGK.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"232 ","pages":"Article 106723"},"PeriodicalIF":1.4,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143906087","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

Enhanced production of functional CRISPR-AsCas12a protein in Escherichia coli 在大肠杆菌中增强功能性CRISPR-AsCas12a蛋白的产生

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-04-25 DOI: 10.1016/j.pep.2025.106722

Orlando S. Goméz-Quintero, Melissa D. Morales-Moreno, Erick G. Valdés-Galindo, Rosa E. Cárdenas-Guerra, Armando Hernandez-Garcia

{"title":"Enhanced production of functional CRISPR-AsCas12a protein in Escherichia coli","authors":"Orlando S. Goméz-Quintero, Melissa D. Morales-Moreno, Erick G. Valdés-Galindo, Rosa E. Cárdenas-Guerra, Armando Hernandez-Garcia","doi":"10.1016/j.pep.2025.106722","DOIUrl":"10.1016/j.pep.2025.106722","url":null,"abstract":"<div><div>The CRISPR-Cas12a system is a groundbreaking tool widely used for genome editing and diagnostics in biotechnology and biomedicine research laboratories. Despite its growing application, studies optimizing Cas12a protein production at the laboratory scale using straightforward protocols remains scarce. This study aimed to enhance the lab-scale recombinant production of <em>Acidaminococcus</em> sp Cas12a protein (AsCas12a) in <em>E. coli</em>. Through targeted adjustments of simple parameters, AsCas12a production was significantly increased. The optimized conditions included the use of <em>E. coli</em> BL21(DE3), TB medium supplemented with 1 % glucose, induction with 0.3 mM IPTG for at least 6–9 h, and incubation at 30 °C. Notably, these conditions differ from conventional protocols typically used for Cas12a and related proteins, such as <em>Streptococcus pyogenes</em> Cas9. Upon combining all optimized parameters, AsCas12a production increased approximately 3-fold, from 0.95 mg/mL of bacterial lysate under non-optimized conditions to 3.73 mg/mL under optimized ones. After chromatographic purification, the final protein yield rose approximately 4.5-fold, from 5.2 to 23.4 mg/L of culture volume, without compromising functional activity. The purified AsCas12a retained full activity for programmable <em>in vitro</em> DNA <em>cis</em>-cleavage and collateral <em>trans</em>-cleavage, which was successfully applied to detect the N gene of SARS-CoV-2. This optimized method provide an efficient and high-yield approach for producing functional AsCas12a protein using accessible materials and conditions available to many research laboratories worldwide.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"232 ","pages":"Article 106722"},"PeriodicalIF":1.4,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143890637","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 recombinant human SLPI variant suppresses the formation of neutrophil extracellular traps at low concentrations in vitro 重组人SLPI变体在体外低浓度抑制中性粒细胞胞外陷阱的形成

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-04-23 DOI: 10.1016/j.pep.2025.106721

Felipe de Jesus Gonzalez-Contreras , Roxana Guadalupe Gutierrez-Vidal , Xristo Zarate

{"title":"A recombinant human SLPI variant suppresses the formation of neutrophil extracellular traps at low concentrations in vitro","authors":"Felipe de Jesus Gonzalez-Contreras , Roxana Guadalupe Gutierrez-Vidal , Xristo Zarate","doi":"10.1016/j.pep.2025.106721","DOIUrl":"10.1016/j.pep.2025.106721","url":null,"abstract":"<div><div>Neutrophil extracellular traps (NETs) are web-like structures released by neutrophils to trap and kill microbes. While NETs are crucial in host defense, excessive formation can lead to autoimmune diseases. Currently, no specific drugs target NETs directly; however, some medications can indirectly modulate their formation. The secretory leukocyte protease inhibitor (SLPI) is a small protein that inhibits the activity of neutrophil elastase (NE), an enzyme essential for NETs formation. By inhibiting NE activity, SLPI prevents the chromatin from decondensing, which is necessary for NETs to form; this suggests that SLPI may protect by preventing excessive NETs development. However, evidence indicates that NE can inactivate SLPI by cleaving its N-terminus. This action can create a protease/antiprotease imbalance, potentially leading to detrimental consequences for the host. In this study, we produced a recombinant variant of SLPI (SLPI-S15G-A16G: rSLPIv) using recombinant DNA technology, expressed in <em>Escherichia coli</em> SHuffle T7. The protein was tagged with the small metal-binding protein (SmbP) to facilitate its expression and purification through immobilized metal-affinity chromatography. Our results demonstrated that rSLPIv exhibited immunomodulatory activity at a concentration of 10 nM in neutrophils that had been prestimulated with PMA. It reduced NETs formation by 30 % and maintained this effect for up to 6 h. Confocal microscopy confirmed these findings, revealing a rSLPIv-dependent reduction in neutrophil nuclear expansion. Thus, rSLPIv shows significant suppressive activity on NETs formation at low concentrations, making it a potential candidate as an immunotherapeutic agent.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"232 ","pages":"Article 106721"},"PeriodicalIF":1.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143898969","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

Biochemical characterization and molecular dynamics study of a thermostable endo-1,5-α-arabinanase 耐热内源性1,5-α-阿拉伯糖酶的生化表征及分子动力学研究

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-04-19 DOI: 10.1016/j.pep.2025.106720

Vandierly Sampaio de Melo , Brisa Moreira Gomes , Tarcillo Gaziri , Elvira Regina Tamarozzi , Felipe Santiago Chambergo

{"title":"Biochemical characterization and molecular dynamics study of a thermostable endo-1,5-α-arabinanase","authors":"Vandierly Sampaio de Melo , Brisa Moreira Gomes , Tarcillo Gaziri , Elvira Regina Tamarozzi , Felipe Santiago Chambergo","doi":"10.1016/j.pep.2025.106720","DOIUrl":"10.1016/j.pep.2025.106720","url":null,"abstract":"<div><div>Arabinan, a crucial constituent of plant biomass, undergoes hydrolysis through the enzymes endo-1,5-α-L-arabinanases and α-L-arabinofuranosidases, resulting in the release of L-arabinose. This monosaccharide holds diverse applications in the biofuel, food, and pharmaceutical industries. In this study, we characterized GeoARA, a recombinant enzyme of endo-1,5-α-L-arabinanase from <em>Geobacillus</em> sp. JS12, previously undescribed in this organism. GeoARA, expressed in <em>E. coli</em> BL21 and purified via affinity chromatography, displayed optimal activity at pH 7.0 and temperature of 70 °C on the specific substrate debranched arabinan. In the presence of metallic ions and EDTA as additives, the enzyme demonstrated high stability. Notably, the enzyme maintained high thermal stability at 70°C for up to 48 h, with enhanced performance in the presence of Co<sup>2+</sup>. GeoARA demonstrated a specific activity on debranched arabinan of 226.7 U mg<sup>−1</sup>, with K<sub>m</sub>, V<sub>max</sub>, k<sub>cat</sub>, and k<sub>cat</sub>/K<sub>m</sub> values of 12.1 mg mL<sup>−1</sup>, 1284.7 μmol min<sup>−1</sup> mg<sup>−1</sup>, 642.3 s<sup>−1</sup>, and 53 mL mg<sup>−1</sup> s<sup>−1</sup>, respectively. Furthermore, the three-dimensional structure of GeoARA was determined using homology-based molecular modeling, and the predicted model quality was validated through molecular dynamics simulations. The integration of computational and biochemical analyses confirmed that the enhanced thermostability observed experimentally, particularly in the presence of the cobalt ion, is associated with reduced atomic fluctuations and increased structural rigidity near the catalytic site. Together, these biochemical features position the endo-1,5-α-L-arabinanase GeoARA as a promising candidate for efficiently extracting L-arabinose in industrial applications.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106720"},"PeriodicalIF":1.4,"publicationDate":"2025-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143852336","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 enzymatic characterization of human UDP-glucose:glycoprotein glucosyltransferase-selenoprotein F complex from Sf9 insect cells Sf9昆虫细胞中人udp -葡萄糖糖蛋白-葡萄糖基转移酶-硒蛋白F复合物的表达、纯化和酶学特性

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-04-16 DOI: 10.1016/j.pep.2025.106719

Hirotaka Tomii , Takashi Kikuma , Hiroyuki Kajiura , Kanae Sano , Hiroyoshi Matsumura , Yukishige Ito , Yasuhiro Kajihara , Yoichi Takeda

{"title":"Expression, purification, and enzymatic characterization of human UDP-glucose:glycoprotein glucosyltransferase-selenoprotein F complex from Sf9 insect cells","authors":"Hirotaka Tomii , Takashi Kikuma , Hiroyuki Kajiura , Kanae Sano , Hiroyoshi Matsumura , Yukishige Ito , Yasuhiro Kajihara , Yoichi Takeda","doi":"10.1016/j.pep.2025.106719","DOIUrl":"10.1016/j.pep.2025.106719","url":null,"abstract":"<div><div>Appropriate functioning of the glycoprotein quality control system in the endoplasmic reticulum is crucial for maintaining cellular homeostasis. UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) is a key component of this system, serving as a “folding sensor\" by recognizing the partially folded state of cognate glycoproteins and reglucosylating their deglucosylated N-glycans. Notably, UGGT1 is known to form heterodimers with Selenoprotein F (SelenoF), although the mechanism underlying the complex formation remains unclear. To date, there have been no reports of large-scale expression systems for recombinant human UGGT1, and the formation of the human UGGT1-SelenoF complex has not been demonstrated <em>in vitro</em>. To address this, we used an insect cell-based expression system for heterologous expression of human UGGT1 and SelenoF, achieving high purity and efficiency superior to that of <em>Escherichia coli</em> expression systems. Importantly, the two proteins, prepared independently, were observed to form complexes <em>in vitro</em>. The establishment of a system for large-scale production of human UGGT1 and UGGT1-SelenoF complexes paves the way for future studies investigating their structural characteristics and dynamic behavior.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106719"},"PeriodicalIF":1.4,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143847898","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-throughput optimization of peptide-linker for fusing function protein with GFP 功能蛋白与绿色荧光蛋白融合肽连接体的高通量优化

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-04-14 DOI: 10.1016/j.pep.2025.106718

Gaili Cao , Zhong Li , Zhaoguan Wang , Youhui Yang , Jiawei Li , Hao Qi

{"title":"High-throughput optimization of peptide-linker for fusing function protein with GFP","authors":"Gaili Cao , Zhong Li , Zhaoguan Wang , Youhui Yang , Jiawei Li , Hao Qi","doi":"10.1016/j.pep.2025.106718","DOIUrl":"10.1016/j.pep.2025.106718","url":null,"abstract":"<div><div>Fusion proteins are pivotal in bioengineering, with applications in purification, delivery, and imaging. However, the development of specialized peptide linkers tailored for target fusion proteins remains an unmet challenge. In this study, we demonstrate the optimization of fusing a functional protein with green fluorescent protein (GFP) through the screening of peptide linker sequences. Using seamless cloning methodology, a nanobody protein was fused to the N-terminus of GFP via a randomized 18-amino acid peptide linker library. Initial screening of fusion protein clones was conducted on solid plates to identify those expressing robust GFP fluorescence. A total of 153 clones with unique linker sequences were identified using Sanger sequencing. A wide range of normalized fluorescence signals was observed, revealing significant variability in linker performance. Among the screened linkers, one exhibited high fluorescence activity, outperforming commonly used flexible and rigid linkers. This finding underscores the necessity of optimize linker sequences for specific fusion proteins. Furthermore, the results demonstrated that the screened linker is compatible with diverse N-terminal proteins while maintaining GFP functionality. Additionally, to investigate the effect of linker on the function of target protein, we determined the reverse transcription efficiency of the murine leukemia virus reverse transcriptase (MLV-RT) in the fusion proteins by a two-step RT-qPCR method. In conclusion, this study presents an efficient optimization of peptide linkers, offering a novel methodology for the engineering and application of specialized linkers for fusion proteins.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"231 ","pages":"Article 106718"},"PeriodicalIF":1.4,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143833615","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