Protein expression and purification最新文献_第9页

Avian primordial germ cell migration: History, mechanisms, applications, and unanswered questions 鸟类原始生殖细胞迁移：历史、机制、应用和未解之谜。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-31 DOI: 10.1016/j.pep.2025.106788

Manami Morimoto , Daisuke Saito

引用次数: 0

Characterisation of ZPH13 phosphotransferase from Bacillus subtilis in zearalenone transformation 枯草芽孢杆菌转化玉米赤霉烯酮ZPH13磷酸转移酶的特性研究

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-22 DOI: 10.1016/j.pep.2025.106783

Yu Zhou , Feng-Jun Qiu , Ju-Bao Li , Yuan-Yuan Zhe , Zi-Xian Cheng , Qian Wu , Hua-Biao Miao , Zun-Xi Huang

{"title":"Characterisation of ZPH13 phosphotransferase from Bacillus subtilis in zearalenone transformation","authors":"Yu Zhou , Feng-Jun Qiu , Ju-Bao Li , Yuan-Yuan Zhe , Zi-Xian Cheng , Qian Wu , Hua-Biao Miao , Zun-Xi Huang","doi":"10.1016/j.pep.2025.106783","DOIUrl":"10.1016/j.pep.2025.106783","url":null,"abstract":"<div><div>Zearalenone (ZEN), a mycotoxin produced by <em>Fusarium</em> species, poses considerable health risks to humans and animals because of its residues in maize and its industrial byproducts. Effective methods to control or eliminate ZEN are urgently needed. Herein, <em>Bacillus subtilis</em> strain Z1Y3, capable of efficient ZEN transformation, was isolated from ZEN-contaminated feed samples. This strain completely transformed 5 mg/L ZEN within 15 h. Subsequently, a ZEN phosphotransferase gene, <em>ZPH13</em>, was identified in its genome. The gene was heterologously expressed in <em>Escherichia coli</em> BL21 (DE3), and the recombinant ZPH13 protein was biochemically characterised. The enzyme exhibited optimal activity at 35 °C and pH 8.0, achieving transformation of 2 μg/mL ZEN within 0.5 h under these conditions. Pythia was used to modify protein stability, identifying potential stabilization hotspots through free energy calculations (ΔΔG) and screening to obtain beneficial mutants N371I and N371T, which improved both enzyme activity and stability. Enzymatic transformation product analysis via ultra-high-performance liquid chromatography–quadrupole exactive–mass spectrometry revealed that ZEN was transformed into zearalenone-phosphate (ZEN-P) via phosphotransferase activity. Furthermore, co-culturing T-47D Human Breast Ductal Carcinoma (T47D) cells with the transformation products demonstrated a considerably reduced toxicity compared to that of untreated ZEN. These findings establish a new paradigm for mycotoxin transformation through innovative enzyme discovery and protein engineering strategies.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106783"},"PeriodicalIF":1.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144695084","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

Modulation of Glutamine Synthetase adenylylation by nitrogen availability enables one-step purification in distinct post-translational states 通过氮可用性调节谷氨酰胺合成酶腺苷化使一步纯化在不同的翻译后状态

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-16 DOI: 10.1016/j.pep.2025.106779

Larissa Fonseca Tomazini , Eduardo Sabatine Lopes , Bárbara Barizão Nogueira , Gabriela Carvalho Calsavara , Ana Paula Santos Silva , Naiara Cristina Lucredi , Edileusa Cristina Marques Gerhardt , Luciano Fernandes Huergo , Marco Aurelio Schuler Oliveira

{"title":"Modulation of Glutamine Synthetase adenylylation by nitrogen availability enables one-step purification in distinct post-translational states","authors":"Larissa Fonseca Tomazini , Eduardo Sabatine Lopes , Bárbara Barizão Nogueira , Gabriela Carvalho Calsavara , Ana Paula Santos Silva , Naiara Cristina Lucredi , Edileusa Cristina Marques Gerhardt , Luciano Fernandes Huergo , Marco Aurelio Schuler Oliveira","doi":"10.1016/j.pep.2025.106779","DOIUrl":"10.1016/j.pep.2025.106779","url":null,"abstract":"<div><div>The glutamine synthetase (GS) enzyme pathway promotes ammonium assimilation in bacteria and is a metabolic hub for glutamine and glutamate homeostasis. Bacterial GS can be reversibly inhibited through adenylylation as a response to nitrogen availability, carried out by the GlnE enzyme. The adenylylation changes GS catalytic and regulatory properties, such as the sensitivity to negative feedback by allosteric modulators and the preferred cofactor usage. In this way, the purification of GS in different modification states can be useful during the investigation of its regulatory properties. Here we show that just by changing nitrogen availability during cell growth it is possible to obtain adenylylated or unmodified GS enzymes after heterologous expression followed by a one-step purification. As a model, we expressed GS enzymes from the diazotrophic bacteria <em>Herbaspirillum seropedicae</em> and <em>Azospirillum brasilense</em> in the M9 media supplemented with ammonium or glutamine. The enzymes were purified by Ni<sup>2+</sup>-affinity chromatography. The data showed that just by varying the nitrogen source during protein expression it was possible to obtain GS in different adenylation status. The different adenylated isoforms of GS obtained were confirmed by electrophoretic mobility shifts and showed unique responses to Mg<sup>2+</sup> and Mn<sup>2+</sup> ions and feedback inhibition by amino acids. Finally, we show the unmodified GS can only bind the glutamate substrate when ATP is present. The method to purify GS on different adenylylation states in a single step described here will facilitate the characterization of this key metabolic enzyme in the future.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106779"},"PeriodicalIF":1.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144655550","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

Scalable approach for coronavirus-like particles making based on the spike protein using Sf9 cells 利用Sf9细胞基于Spike蛋白制造冠状病毒样颗粒的可扩展方法。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-03 DOI: 10.1016/j.pep.2025.106767

Luis Giovani de Oliveira Guardalini , Thaissa Consoni Bernardino , Felipe Moura Dias , Jaci Leme , Henrique Coelho de Oliveira , Aldo Tonso , Eutimio Gustavo Fernández Núñez , Soraia Attie Calil Jorge

{"title":"Scalable approach for coronavirus-like particles making based on the spike protein using Sf9 cells","authors":"Luis Giovani de Oliveira Guardalini , Thaissa Consoni Bernardino , Felipe Moura Dias , Jaci Leme , Henrique Coelho de Oliveira , Aldo Tonso , Eutimio Gustavo Fernández Núñez , Soraia Attie Calil Jorge","doi":"10.1016/j.pep.2025.106767","DOIUrl":"10.1016/j.pep.2025.106767","url":null,"abstract":"<div><div>The COVID-19 pandemic has led to a race to find prevention methods, such as vaccines. Virus-like particles (VLP), mimicking native viruses without genetic material, represent a promising vaccine platform due to their safety and strong immunogenicity. The baculovirus expression system, using insect cells like Sf9, offers an efficient method for VLP production. This study investigated the coinfection of Sf9 cells with recombinant baculoviruses carrying genes for SARS-CoV-2 spike, membrane, nucleocapsid, and envelope proteins in stirred tank bioreactors. Spike protein expression, VLP formation, and cellular metabolic profiles were analyzed. Bioreactor assays revealed suitable conditions for VLP production, demonstrating the robustness of this platform to handle emerging viral variants. These findings support the large-scale production of VLP carrying spike protein as the main immunogen as a versatile platform for vaccine development against SARS-CoV-2 and other coronaviruses and future viral threats, enabling a rapid response to emerging outbreaks with few changes.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106767"},"PeriodicalIF":1.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144567752","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

Rapid purification and characterization of angiotensin I-converting enzyme from porcine lung 猪肺血管紧张素i转化酶的快速纯化及特性研究。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-17 DOI: 10.1016/j.pep.2025.106780

Meng Li , Pan-Pan Xiao , Wan-Yu Li , Ling-Jing Zhang , Yu-Lei Chen , Tengchuan Jin , Min-Jie Cao

{"title":"Rapid purification and characterization of angiotensin I-converting enzyme from porcine lung","authors":"Meng Li , Pan-Pan Xiao , Wan-Yu Li , Ling-Jing Zhang , Yu-Lei Chen , Tengchuan Jin , Min-Jie Cao","doi":"10.1016/j.pep.2025.106780","DOIUrl":"10.1016/j.pep.2025.106780","url":null,"abstract":"<div><div>Angiotensin I-converting enzyme (ACE) is a key target for screening hypertension medicines. However, traditional method for ACE preparation is time-consuming, and commercial ACE is expensive. In this study, a simple and effective method for ACE purification was proposed. ACE was purified to homogeneity from porcine lungs using acid precipitation, ammonium sulfate fractionation, and chromatography on a HiTrap Q HP column. 2D-PAGE showed that the molecular weight of ACE was 180 kDa, and the p<em>I</em> was 5.7, its sequence was verified by LC-MS/MS. The thermal denaturation temperature of ACE was 58.8 ± 0.4 °C and it is a glycoprotein as confirmed by PAS staining. Circular dichroism and endogenous fluorescence spectroscopy showed that the addition of zinc ions led to changes in the structure of ACE, thereby inhibiting its activity. Compared with commercial ACE, the enzyme prepared in the present study exhibited higher purity and 1.3-fold higher specific activity. As ACE is widely required for studies on anti-hypertensive functional foods, effective preparation of high-purity ACE will be valuable for the investigation of potential functional foods and especially its inhibitory peptides.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106780"},"PeriodicalIF":1.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144668223","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

Functional expression of five refolded recombinant variants of RBD from SARS-CoV-2 in Escherichia coli 五种重组SARS-CoV-2 RBD变体在大肠杆菌中的功能表达

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-17 DOI: 10.1016/j.pep.2025.106781

Ezequiel Tobón , Lidia Riaño-Umbarila , Humberto Flores , Michelle Gutierrez , Xavier Soberón , Baltazar Becerril-Lujan , Octavio T. Ramírez , Enrique Rudiño-Piñera

{"title":"Functional expression of five refolded recombinant variants of RBD from SARS-CoV-2 in Escherichia coli","authors":"Ezequiel Tobón , Lidia Riaño-Umbarila , Humberto Flores , Michelle Gutierrez , Xavier Soberón , Baltazar Becerril-Lujan , Octavio T. Ramírez , Enrique Rudiño-Piñera","doi":"10.1016/j.pep.2025.106781","DOIUrl":"10.1016/j.pep.2025.106781","url":null,"abstract":"<div><div>Production of the receptor binding domain, RBD, variants of SARS-CoV-2, has been necessary to understand their interaction with the human receptor ACE2 and to develop alternative strategies against COVID-19. Affordable bacterial production can aid in these efforts and offer alternatives to address the ongoing emergence of new variants. Expressing recombinant RBD in <em>E. coli</em> could provide a valuable alternative if the challenges related to disulfide bond formation, low solubility, and the absence of glycosylation capabilities are addressed in this heterologous expression system. In this study, five representative RBD variants were expressed in <em>E. coli</em> BL21 (two versions of the RBD Wuhan sequence (Wt), Delta, Omicron BA.2, and Omicron JN.1). The resulting inclusion bodies were solubilized using guanidine chloride and refolded in the presence of a buffer containing the redox couple GSH-GSSG and L-arginine monohydrochloride. Purification was achieved through size-exclusion chromatography. These purified RBD variants were analyzed and compared to the RBD with the Wuhan sequence and the Omicron BA.5 variant, both produced in Chinese Hamster Ovary cells, regarding their ability to bind to ACE2 and using surface plasmon resonance, SPR, studies. The strategy outlined in this work did not include expression studies or a complete comparison of the expression yields obtained in relation to other expression systems. However, it clearly demonstrates the feasibility of obtaining various RBD variants, capable of binding to ACE2, starting from inclusion bodies, and exhibiting comparable affinities to those RBD variants produced in eukaryotic cells. This approach provides an additional option to enhance the existing tools available for research laboratories studying interactions between biologically active RBD variants and ACE2.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106781"},"PeriodicalIF":1.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144668222","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

Scalable production of functional recombinant human plasma gelsolin in Escherichia coli for therapeutic and diagnostic applications 在大肠杆菌中大规模生产用于治疗和诊断的功能性重组人血浆凝胶。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-30 DOI: 10.1016/j.pep.2025.106786

Young Su Kim , Hye-Jeong Lee , Mi-Reu Kim , Hwabong Jeong , Young Pil Kim , Jung-Ho Park , Jungoh Ahn

{"title":"Scalable production of functional recombinant human plasma gelsolin in Escherichia coli for therapeutic and diagnostic applications","authors":"Young Su Kim , Hye-Jeong Lee , Mi-Reu Kim , Hwabong Jeong , Young Pil Kim , Jung-Ho Park , Jungoh Ahn","doi":"10.1016/j.pep.2025.106786","DOIUrl":"10.1016/j.pep.2025.106786","url":null,"abstract":"<div><div>Human plasma gelsolin (pGSN) is an 83 kDa actin-binding protein involved in cytoskeletal remodeling, inflammation, and host defense. Its clinical relevance as a biomarker and potential therapeutic agent, particularly in conditions like sepsis, acute respiratory distress syndrome (ARDS), and cystic fibrosis, has driven interest in scalable recombinant expression. However, high-yield production of functionally active gelsolin is hindered by its complex structure and folding requirements. To address this, we developed a scalable, high-yield bacterial expression system that achieves among the highest reported levels of functional recombinant human gelsolin (rGelsolin) using a GST-fusion strategy incorporating a tobacco etch virus (TEV) protease cleavage site, optimized for solubility and downstream processing. High-density fed-batch fermentation in <em>E. coli</em> yielded 5.0 g/L of soluble protein. Following a three-step purification process with removal of the GST tag, 2.1 g/L of tag-free, high-purity rGelsolin with >95 % purity was obtained. Structural characterization by circular dichroism spectroscopy confirmed that rGelsolin adopted a native-like secondary structure and exhibited thermal stability (Tm ∼59 °C). Correct processing of the recombinant protein was verified by N- and C-terminal sequencing. Functional assays demonstrated that rGelsolin bound to and severed actin filaments in a calcium-dependent manner, similar to native plasma gelsolin. These findings demonstrate a scalable, cost-effective platform for producing bioactive rGelsolin in <em>E. coli</em>, with structural and functional features comparable to native pGSN, supporting its potential utility in diagnostic, therapeutic, and structural applications in the context of acute and chronic inflammatory diseases.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106786"},"PeriodicalIF":1.2,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144765319","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

Ligand multimerization effects on binding efficiency of Protein L affinity chromatography resins 配体多聚对蛋白L亲和层析树脂结合效率的影响

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-08-05 DOI: 10.1016/j.pep.2025.106790

Jafar Nikzad , Kimia Kalantari Khandani , Yeganeh Talebkhan , Fatemeh Zandi , Samira Komijani , Ahmad Adeli

{"title":"Ligand multimerization effects on binding efficiency of Protein L affinity chromatography resins","authors":"Jafar Nikzad , Kimia Kalantari Khandani , Yeganeh Talebkhan , Fatemeh Zandi , Samira Komijani , Ahmad Adeli","doi":"10.1016/j.pep.2025.106790","DOIUrl":"10.1016/j.pep.2025.106790","url":null,"abstract":"<div><div>Nearly four decades have passed since the discovery of protein-L. Its exceptional ability in binding to the kappa light chain of immunoglobulins makes it a suitable candidate for the purification of certain biotherapeutics, particularly antibody fragments. Efforts have focused on improving its recovery and dynamic binding capacity. Among various strategies, ligand multimerization has shown significant potential in developing more efficient and cost-effective resins. This study employed a multimerization approach to create new recombinant protein-L-based ligands and compare their performance to the commercially available alternatives. Dynamic binding capacity studies revealed that the engineered ProL6 and ProL8 resins exhibited higher binding capacities than the ProL4 and the commercial Capto-L resin. Furthermore, the recovery rates of Fab antibody fragments from bacterial lysates using ProL6, ProL8, and the commercial resin were 92.8, 94.4, and 94.6 %, respectively, comparable to those of the ProL4 resin. SDS-PAGE analysis confirmed the purity of the proteins eluted from all tested resins, aligned with the results of ProL4 resin. Additionally, it evaluated the 100 % specificity of ProL6, ProL8, and the commercial resins.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106790"},"PeriodicalIF":1.2,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144770612","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 : 2025-10-01 Epub 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":"2025-10-01","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

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 : 2025-10-01 Epub 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":"2025-10-01","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