Protein expression and purification最新文献_第2页

Expression, purification and characterization of a novel triple fusion protein developed for the immunotherapy of survivin positive cancers 用于存活素阳性癌症免疫疗法的新型三重融合蛋白的表达、纯化和表征。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-10-11 DOI: 10.1016/j.pep.2024.106614

Ambreen Rashid , Mohammad Azad , Anuja Krishnan , Jagdish C. Gupta , G.P. Talwar

Survivin is an inhibitor of apoptosis, and expressed in a large number of cancers. As Survivin expression is very low in normal tissues, it assumes significance as a prominent target for tumor diagnosis, prognosis and developing anti-cancer therapies. We report development of a novel triple fusion protein for a prospective vaccine against Survivin in targeted cancer immunotherapy. A gene was synthesized by combining the nucleotides encoding human origin Survivin and heat-labile enterotoxin of Escherichia coli (LTB). Further, nucleotides corresponding to single chain variable fragment (scFv) of a monoclonal having affinity for DEC205 receptor present on dendritic cells, were also incorporated into the gene sequence. This complete gene was expressed to a triple fusion recombinant protein using a bacterial expression vector under the control of robust bacteriophage T7 promoter. The recombinant _DCSurvivin-LTB protein, with a size of approximately 60 kDa, was purified from the inclusion bodies using affinity based Ni-NTA columns. The purified protein was confirmed by the Western blot, and further characterized with circular dichroism, fluorescence spectroscopy and mass spectroscopy. This molecularly adjuvanted Survivin fusion protein designed to deliver to the dendritic cells for better antigen processing, elicited a stronger anti-Survivin immune response compared to Survivin protein alone. It can be an effective vaccine in active and passive immunotherapies for Survivin expressing cancer cells.

Survivin 是一种细胞凋亡抑制因子，在许多癌症中都有表达。由于 Survivin 在正常组织中的表达量很低，因此它成为肿瘤诊断、预后和开发抗癌疗法的重要靶点。我们报告了一种新型三重融合蛋白的开发情况，该蛋白可用于靶向癌症免疫疗法中的 Survivin 疫苗。我们将编码人源 Survivin 和大肠杆菌热嗜性肠毒素（LTB）的核苷酸合成了一个基因。此外，基因序列中还加入了与树突状细胞上 DEC205 受体具有亲和力的单克隆的单链可变片段（scFv）相对应的核苷酸。在强健的噬菌体 T7 启动子控制下，使用细菌表达载体将这一完整基因表达为三重融合重组蛋白。重组的 DCSurvivin-LTB 蛋白大小约为 60 kDa，使用亲和基 Ni-NTA 柱从包涵体中纯化出来。纯化的蛋白质经 Western 印迹法确认，并通过圆二色性、荧光光谱和质谱法进一步鉴定。与单独的 Survivin 蛋白相比，这种分子佐剂化的 Survivin 融合蛋白能激发更强的抗 Survivin 免疫反应。在针对表达 Survivin 的癌细胞的主动和被动免疫疗法中，它可以成为一种有效的疫苗。

{"title":"Expression, purification and characterization of a novel triple fusion protein developed for the immunotherapy of survivin positive cancers","authors":"Ambreen Rashid , Mohammad Azad , Anuja Krishnan , Jagdish C. Gupta , G.P. Talwar","doi":"10.1016/j.pep.2024.106614","DOIUrl":"10.1016/j.pep.2024.106614","url":null,"abstract":"<div><div>Survivin is an inhibitor of apoptosis, and expressed in a large number of cancers. As Survivin expression is very low in normal tissues, it assumes significance as a prominent target for tumor diagnosis, prognosis and developing anti-cancer therapies. We report development of a novel triple fusion protein for a prospective vaccine against Survivin in targeted cancer immunotherapy. A gene was synthesized by combining the nucleotides encoding human origin Survivin and heat-labile enterotoxin of Escherichia coli (LTB). Further, nucleotides corresponding to single chain variable fragment (scFv) of a monoclonal having affinity for DEC205 receptor present on dendritic cells, were also incorporated into the gene sequence. This complete gene was expressed to a triple fusion recombinant protein using a bacterial expression vector under the control of robust bacteriophage T7 promoter. The recombinant DCSurvivin-LTB protein, with a size of approximately 60 kDa, was purified from the inclusion bodies using affinity based Ni-NTA columns. The purified protein was confirmed by the Western blot, and further characterized with circular dichroism, fluorescence spectroscopy and mass spectroscopy. This molecularly adjuvanted Survivin fusion protein designed to deliver to the dendritic cells for better antigen processing, elicited a stronger anti-Survivin immune response compared to Survivin protein alone. It can be an effective vaccine in active and passive immunotherapies for Survivin expressing cancer cells.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106614"},"PeriodicalIF":1.4,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142473255","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

N-linked glycosylation affects catalytic parameters and fluctuation of the active center of Aspergillus awamori exo-inulinase N-连接的糖基化影响黑曲霉 awamori 外胰岛素酶活性中心的催化参数和波动。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-09-30 DOI: 10.1016/j.pep.2024.106613

Anna Dotsenko , Yury Denisenko , Ivan Zorov , Aleksandra Rozhkova , Igor Shashkov

Heterogeneous expression of enzymes allows large-scale production with reduced costs. Changes in glycosylation often occur due to changes in the expression host. In the study, the catalytic and biochemical properties of Aspergillus awamori exo-inulinase 1 are compared for A. awamori and Penicillium verruculosum expression hosts. The tertiary structure contains seven sites of N-glycosylation, with two of them located near the active center. If expressed in P. verruculosum, the enzyme was four times less glycosylated and two times more active toward sucrose, raffinose, and stachyose due to an increase in k_cat. These substrates with a short chain of 2–4 monosaccharide units were used to characterize the interaction of the substrate with the amino acid residues in the active center while preventing the interaction of the substrate with N-linked glycans. Molecular dynamics simulations showed an increase in the fluctuation of the active center with an increase in the length of N-linked glycans. The fluctuation of the residues N40 and Q57, which interact with the hydroxyl group O5 of the fructose unit in the −1 subsite of the active center, was increased by 1.6 times. The fluctuation of the residue W335, which interacts with the hydroxyl group O1 of the fructose unit together with the catalytic residue D41 and affects the torsion angle geometry of the substrate molecules, was increased by 1.5 times. The residue R188, which analogously to W335 affects the torsion angle geometry of the substrate molecules, was also among the affected residues with a 1.2-fold increase in the fluctuation.

酶的异构表达可以大规模生产，降低成本。糖基化的变化往往是由表达宿主的变化引起的。在这项研究中，比较了阿瓦莫里曲霉外胰岛素酶 1 在阿瓦莫里曲霉和疣青霉表达宿主下的催化和生化特性。其三级结构包含七个 N-糖基化位点，其中两个位于活性中心附近。如果在疣青霉中表达，由于 kcat 的增加，该酶的糖基化程度降低四倍，对蔗糖、棉子糖和水苏糖的活性提高两倍。这些底物具有 2-4 个单糖单位的短链，用于表征底物与活性中心氨基酸残基的相互作用，同时防止底物与连接糖的相互作用。分子动力学模拟显示，活性中心的波动会随着 N 联糖类长度的增加而增加。残基 N40 和 Q57 与活性中心 -1 亚位点果糖单元的羟基 O5 相互作用，其波动增加了 1.6 倍。与催化残基 D41 一起与果糖单元的羟基 O1 相互作用并影响底物分子扭转角几何形状的残基 W335 的波动增加了 1.5 倍。与 W335 类似影响底物分子扭转角几何形状的残基 R188 也是受影响的残基之一，其波动增加了 1.2 倍。

{"title":"N-linked glycosylation affects catalytic parameters and fluctuation of the active center of Aspergillus awamori exo-inulinase","authors":"Anna Dotsenko , Yury Denisenko , Ivan Zorov , Aleksandra Rozhkova , Igor Shashkov","doi":"10.1016/j.pep.2024.106613","DOIUrl":"10.1016/j.pep.2024.106613","url":null,"abstract":"<div><div>Heterogeneous expression of enzymes allows large-scale production with reduced costs. Changes in glycosylation often occur due to changes in the expression host. In the study, the catalytic and biochemical properties of Aspergillus awamori exo-inulinase 1 are compared for A. awamori and Penicillium verruculosum expression hosts. The tertiary structure contains seven sites of N-glycosylation, with two of them located near the active center. If expressed in P. verruculosum, the enzyme was four times less glycosylated and two times more active toward sucrose, raffinose, and stachyose due to an increase in kcat. These substrates with a short chain of 2–4 monosaccharide units were used to characterize the interaction of the substrate with the amino acid residues in the active center while preventing the interaction of the substrate with N-linked glycans. Molecular dynamics simulations showed an increase in the fluctuation of the active center with an increase in the length of N-linked glycans. The fluctuation of the residues N40 and Q57, which interact with the hydroxyl group O5 of the fructose unit in the −1 subsite of the active center, was increased by 1.6 times. The fluctuation of the residue W335, which interacts with the hydroxyl group O1 of the fructose unit together with the catalytic residue D41 and affects the torsion angle geometry of the substrate molecules, was increased by 1.5 times. The residue R188, which analogously to W335 affects the torsion angle geometry of the substrate molecules, was also among the affected residues with a 1.2-fold increase in the fluctuation.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106613"},"PeriodicalIF":1.4,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142366345","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

Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana: Characterization of the enzyme's in vitro truncation by thiol endopeptidase activity 从模式植物拟南芥中异源表达和纯化谷氨酸脱羧酶-1：通过硫醇内肽酶活性鉴定体外截短酶的特性。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-09-27 DOI: 10.1016/j.pep.2024.106612

Brittany S. Menard , Kirsten H. Benidickson , Lee Marie Raytek , Wayne A. Snedden , William C. Plaxton

Plant glutamate decarboxylase (GAD) is a Ca²⁺-calmodulin (CaM) activated enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. Our prior research established that in vivo phosphorylation of AtGAD1 (AT5G17330) occurs at multiple N-terminal serine residues following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 (rAtGAD1) following its expression in Escherichia coli to facilitate studies of the impact of phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of an approximate 5 kDa polypeptide from the C-terminus of 59 kDa rAtGAD1 subunits occurred during purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing this partial rAtGAD1 proteolysis. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated rAtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalysis, and that rAtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca²⁺/CaM facilitated the purification of non-proteolyzed rAtGAD1 to a specific activity of 27 (μmol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca²⁺/CaM at pH 7.3. By contrast, the purified partially proteolyzed rAtGAD1 was >40 % less active at both pH values, and only activated 2-fold by Ca²⁺/CaM at pH 7.3. These results emphasize the need to diagnose and prevent partial proteolysis before conducting kinetic studies of purified regulatory enzymes.

植物谷氨酸脱羧酶（GAD）是一种由 Ca2+ -钙调蛋白（CaM）激活的酶，它产生γ-氨基丁酸（GABA），是 GABA 分流的第一步。我们之前的研究证实，向模式植物拟南芥的缺∏细胞培养物重新补充∏后，AtGAD1（AT5G17330）的多个 N 端丝氨酸残基会发生体内磷酸化。目前研究的目的是在大肠杆菌中表达重组 AtGAD1（rAtGAD1）后对其进行纯化，以便研究磷酸化对其动力学特性的影响。然而，在纯化过程中，59 kDa rAtGAD1 亚基 C 端约 5 kDa 的多肽发生了体外蛋白水解截短。免疫印迹表明，我们测试过的大多数蛋白酶抑制剂或混合抑制剂都不能有效抑制这种部分 rAtGAD1 蛋白水解。虽然硫醇修饰剂 N-乙基马来酰亚胺或 2,2-二吡啶基二硫化物能抑制 rAtGAD1 的蛋白水解，但它们也会削弱其 GAD 活性。这表明催化作用需要一个重要的 -SH 基团，而且 rAtGAD1 易于被大肠杆菌半胱氨酸内肽酶或可能通过自体蛋白水解活性部分降解。加入外源 Ca2+/CaM 有助于纯化未被蛋白水解的 rAtGAD1，在最佳 pH 值为 5.8 时，rAtGAD1 的特异性活性为 27（产生的 GABA μmol/mg），而在 pH 值为 7.3 时，Ca2+/CaM 可将其激活约 3 倍。相比之下，纯化的部分蛋白水解的 rAtGAD1 在两种 pH 值下的活性都要低 40%，在 pH 7.3 时仅被 Ca2+/CaM 激活 2 倍。这些结果表明，在对纯化的调节酶进行动力学研究之前，需要诊断和防止部分蛋白水解。

{"title":"Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana: Characterization of the enzyme's in vitro truncation by thiol endopeptidase activity","authors":"Brittany S. Menard , Kirsten H. Benidickson , Lee Marie Raytek , Wayne A. Snedden , William C. Plaxton","doi":"10.1016/j.pep.2024.106612","DOIUrl":"10.1016/j.pep.2024.106612","url":null,"abstract":"<div><div>Plant glutamate decarboxylase (GAD) is a Ca2+-calmodulin (CaM) activated enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. Our prior research established that in vivo phosphorylation of AtGAD1 (AT5G17330) occurs at multiple N-terminal serine residues following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 (rAtGAD1) following its expression in Escherichia coli to facilitate studies of the impact of phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of an approximate 5 kDa polypeptide from the C-terminus of 59 kDa rAtGAD1 subunits occurred during purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing this partial rAtGAD1 proteolysis. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated rAtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalysis, and that rAtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca2+/CaM facilitated the purification of non-proteolyzed rAtGAD1 to a specific activity of 27 (μmol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca2+/CaM at pH 7.3. By contrast, the purified partially proteolyzed rAtGAD1 was >40 % less active at both pH values, and only activated 2-fold by Ca2+/CaM at pH 7.3. These results emphasize the need to diagnose and prevent partial proteolysis before conducting kinetic studies of purified regulatory enzymes.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106612"},"PeriodicalIF":1.4,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142352672","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Evaluation of BVDV E2 proteins based on recombinant baculovirus expression system production as diagnostic antigens and immunogens 基于重组杆状病毒表达系统生产的 BVDV E2 蛋白作为诊断抗原和免疫原的评估

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-09-22 DOI: 10.1016/j.pep.2024.106611

Jinke He , Xiaoyu Deng , Xusheng Ma , Liangjia Yao , Yiguo Li , Chuangfu Chen , Yanhua He

Bovine viral diarrhea virus (BVDV) is a significant immunosuppressive pathogen that has a major impact on the global cattle industry. Research efforts are currently focused on the envelope glycoprotein E2 of BVDV to improve immune responses. However, the full-length E2 protein is not ideal as an immune antigen and diagnostic tool, leading to the exploration of alternative strategies. In this study, we optimized the E2 gene using IDEB and ExpOptimizer software, then expressed the E2 gene using both baculovirus and E. coli expression systems. Subsequently, we assessed the immunogenicity of the purified E2 protein in mice and its application in indirect ELISA assays. Our findings showed that the Bac-E2 protein produced by the baculovirus system induced higher levels of antibody production and splenic lymphocyte proliferation in mice compared to the E. coli system. Moreover, the indirect ELISA assay developed using Bac-E2 protein exhibited superior specificity, sensitivity, and accuracy in comparison to the E. coli-expressed E2 ELISA. Overall, our study demonstrates that the optimized E2 protein generated through a baculovirus expression system elicits robust humoral and cellular immune responses in mice, making it a promising candidate for vaccine development. Furthermore, the optimized E2 protein ELISA assay shows enhanced sensitivity and accuracy, suggesting its potential as a valuable diagnostic antigen.

牛病毒性腹泻病毒（BVDV）是一种对全球养牛业有重大影响的重要免疫抑制病原体。目前，研究工作主要集中在 BVDV 的包膜糖蛋白 E2 上，以改善免疫反应。然而，全长 E2 蛋白作为免疫抗原和诊断工具并不理想，因此需要探索替代策略。在本研究中，我们使用 IDEB 和 ExpOptimizer 软件优化了 E2 基因，然后使用杆状病毒和大肠杆菌两种表达系统表达了 E2 基因。随后，我们评估了纯化的 E2 蛋白在小鼠体内的免疫原性及其在间接 ELISA 检测中的应用。我们的研究结果表明，与大肠杆菌系统相比，由杆状病毒系统产生的 Bac-E2 蛋白能诱导小鼠产生更高水平的抗体和脾脏淋巴细胞增殖。此外，与大肠杆菌表达的E2 ELISA相比，利用Bac-E2蛋白开发的间接ELISA检测法在特异性、灵敏度和准确性方面都更胜一筹。总之，我们的研究表明，通过杆状病毒表达系统生成的优化 E2 蛋白可在小鼠体内引起强大的体液和细胞免疫反应，因此有望成为疫苗开发的候选物质。此外，优化后的 E2 蛋白 ELISA 检测法显示出更高的灵敏度和准确性，这表明它有潜力成为一种有价值的诊断抗原。

{"title":"Evaluation of BVDV E2 proteins based on recombinant baculovirus expression system production as diagnostic antigens and immunogens","authors":"Jinke He , Xiaoyu Deng , Xusheng Ma , Liangjia Yao , Yiguo Li , Chuangfu Chen , Yanhua He","doi":"10.1016/j.pep.2024.106611","DOIUrl":"10.1016/j.pep.2024.106611","url":null,"abstract":"<div><div>Bovine viral diarrhea virus (BVDV) is a significant immunosuppressive pathogen that has a major impact on the global cattle industry. Research efforts are currently focused on the envelope glycoprotein E2 of BVDV to improve immune responses. However, the full-length E2 protein is not ideal as an immune antigen and diagnostic tool, leading to the exploration of alternative strategies. In this study, we optimized the E2 gene using IDEB and ExpOptimizer software, then expressed the E2 gene using both baculovirus and E. coli expression systems. Subsequently, we assessed the immunogenicity of the purified E2 protein in mice and its application in indirect ELISA assays. Our findings showed that the Bac-E2 protein produced by the baculovirus system induced higher levels of antibody production and splenic lymphocyte proliferation in mice compared to the E. coli system. Moreover, the indirect ELISA assay developed using Bac-E2 protein exhibited superior specificity, sensitivity, and accuracy in comparison to the E. coli-expressed E2 ELISA. Overall, our study demonstrates that the optimized E2 protein generated through a baculovirus expression system elicits robust humoral and cellular immune responses in mice, making it a promising candidate for vaccine development. Furthermore, the optimized E2 protein ELISA assay shows enhanced sensitivity and accuracy, suggesting its potential as a valuable diagnostic antigen.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106611"},"PeriodicalIF":1.4,"publicationDate":"2024-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142314941","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

Anti-biofilm and antibacterial effect of bacteriocin derived from Lactobacillus plantarum on the multidrug-resistant Acinetobacter baumannii 植物乳杆菌提取的细菌素对耐多药鲍曼不动杆菌的抗生物膜和抗菌作用

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-09-19 DOI: 10.1016/j.pep.2024.106610

Kasra Javadi , Mohammad Reza Emadzadeh , Seyed Amir Hossein Mohammadzadeh Hosseini Moghri , Mehrdad Halaji , Hadi Parsian , Mehdi Rajabnia , Abazar Pournajaf

This research examines the impact of bacteriocin derived from Lactobacillus plantarum PTCC 1745 on the biofilm formations of A. baumannii isolates. Bacteriocin derived from L. plantarum PTCC 1745 was obtained through ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Testing for bacteriocin susceptibility has been conducted using the broth dilution method. The anti-biofilm activity of bacteriocin was evaluated using a microtiter plate method. Quantitative real-time PCR assay evaluated bap gene expression in bacteriocin-treated cells. According to SDS-PAGE, bacteriocin from L. plantarum has a 25-kDa apparent molecular weight. The MICs of bacteriocin ranged from 30 to 120 μg/mL, while the MBCs varied between 60 and 120 μg/mL. Compared to the non-treated group, strains bacteriocin-treated isolates had 59 % less ability to form biofilm. The mean relative expression of the bap gene among the MDR A. baumannii isolates decreased by 52 % compared to the untreated control. This study demonstrated that bacteriocin derived from L. plantarum PTCC 1745 had antibacterial and antibiofilm activity against MDR A. baumannii isolates.

本研究探讨了植物乳杆菌（Lactobacillus plantarum PTCC 1745）提取的细菌素对鲍曼尼氏菌分离株生物膜形成的影响。通过硫酸铵沉淀、阳离子交换色谱法和反相高效液相色谱法（RP-HPLC）获得了植物乳杆菌 PTCC 1745 的细菌素。细菌素敏感性测试采用肉汤稀释法进行。细菌素的抗生物膜活性采用微孔板法进行评估。实时定量 PCR 检测评估了细菌素处理过的细胞中 bap 基因的表达。根据 SDS-PAGE 分析，植物桿菌细菌素的表观分子量为 25-kDa。细菌素的 MIC 在 30 至 120 μg/mL 之间，而 MBC 在 60 至 120 μg/mL 之间。与未处理组相比，经细菌素处理的菌株形成生物膜的能力降低了 59%。与未处理的对照组相比，MDR鲍曼不动杆菌分离株中bap基因的平均相对表达量减少了52%。本研究表明，植物酵母菌 PTCC 1745 衍生的细菌素对 MDR 鲍曼尼氏菌分离株具有抗菌和抗生物膜活性。

{"title":"Anti-biofilm and antibacterial effect of bacteriocin derived from Lactobacillus plantarum on the multidrug-resistant Acinetobacter baumannii","authors":"Kasra Javadi , Mohammad Reza Emadzadeh , Seyed Amir Hossein Mohammadzadeh Hosseini Moghri , Mehrdad Halaji , Hadi Parsian , Mehdi Rajabnia , Abazar Pournajaf","doi":"10.1016/j.pep.2024.106610","DOIUrl":"10.1016/j.pep.2024.106610","url":null,"abstract":"<div><div>This research examines the impact of bacteriocin derived from Lactobacillus plantarum PTCC 1745 on the biofilm formations of A. baumannii isolates. Bacteriocin derived from L. plantarum PTCC 1745 was obtained through ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Testing for bacteriocin susceptibility has been conducted using the broth dilution method. The anti-biofilm activity of bacteriocin was evaluated using a microtiter plate method. Quantitative real-time PCR assay evaluated bap gene expression in bacteriocin-treated cells. According to SDS-PAGE, bacteriocin from L. plantarum has a 25-kDa apparent molecular weight. The MICs of bacteriocin ranged from 30 to 120 μg/mL, while the MBCs varied between 60 and 120 μg/mL. Compared to the non-treated group, strains bacteriocin-treated isolates had 59 % less ability to form biofilm. The mean relative expression of the bap gene among the MDR A. baumannii isolates decreased by 52 % compared to the untreated control. This study demonstrated that bacteriocin derived from L. plantarum PTCC 1745 had antibacterial and antibiofilm activity against MDR A. baumannii isolates.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106610"},"PeriodicalIF":1.4,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142293976","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Cloning and characterization of a novel nitric oxide synthase gene from Exiguobacterium profundum and its expression in Escherichia coli 一氧化氮合成酶基因的克隆和特性鉴定及其在大肠杆菌中的表达。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-09-18 DOI: 10.1016/j.pep.2024.106609

Xifan Luo, Xinyu Li, Yaru Zhang, Fei Zhao, Jinlong Wang, Jiang Wu

The recognition and characterization of gene-encoded nitric oxide synthase (NOS) from Exiguobacterium profundum are reported in this study. A new gene was sequenced and cloned from E. profundum and heterologously expressed in E. coli for functional identification, followed by protein purification using the His-tag. The stability and activity characteristics of the recombinant NOS were evaluated using different concentrations of IPTG at various time points. A band of approximately 42 kDa was observed by SDS-PAGE. The K_m value of NOS, calculated based on the Michaelis-Menten equation was 0.59 μmol/L. Additionally, homologous sequence alignment analysis indicated that the new NOS shared 80.48 % similarity with the same protein from Bacillus subtilis and Umezawaea. The construction of the NOS expression vector and the purification of the recombinant protein provide a foundation for further functional research and inhibitor development.

本研究报告了对深海外杆菌基因编码的一氧化氮合酶（NOS）的识别和表征。研究人员对深沟外杆菌的一个新基因进行了测序和克隆，并在大肠杆菌中进行了异源表达以进行功能鉴定，随后使用 His 标记对蛋白质进行了纯化。在不同时间点使用不同浓度的 IPTG 对重组 NOS 的稳定性和活性特征进行了评估。通过 SDS-PAGE 可以观察到一条大约 42 kDa 的条带。根据 Michaelis-Menten 方程计算，NOS 的 Km 值为 0.59 μmol/L。此外，同源序列比对分析表明，新的 NOS 与枯草芽孢杆菌和梅泽藻中的相同蛋白有 80.48% 的相似性。NOS表达载体的构建和重组蛋白的纯化为进一步的功能研究和抑制剂开发奠定了基础。

引用次数: 0

Characterization of a marine endolysin LysVPB against Vibrio parahaemolyticus 抗副溶血性弧菌的海洋内溶菌素 LysVPB 的特征

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-09-16 DOI: 10.1016/j.pep.2024.106608

Juan Chen , Ziyun Zhao , Xiaofeng Mu , Mengxin Wang , Jun Tang , Qingqing Bi

Currently, there is an urgent to develop safe and environmentally friendly alternatives to antibiotics for combating Vibrio parahaemolyticus. Endolysins are considered promising antibacterial agents due to their desirable range of action and ability to deal with antibiotic-resistant bacteria. While numerous Vibrio phages have been identified, the research on their endolysins is still in its infancy. In this study, a novel endolysin called LysVPB was cloned and expressed in Pichia pastoris. Phylogenetic analysis revealed that LysVPB bears little resemblance to other known endolysins, highlighting its unique nature. Homology modeling identified a putative calcium-binding site in LysVPB. The recombinant LysVPB achieved a lytic activity of 64.8 U/mL and had a molecular weight of approximately 17 kDa. LysVPB exhibited enhanced efficacy at pH 9.0, with 60 % of its maximum activity observed within the broad pH range of 6.0–10.0. The catalytic efficiency of LysVPB peaked at 30 °C but significantly declined beyond 50 °C. Ba²⁺, Co²⁺, and Cu²⁺ showed inhibitory effects on the activity of LysVPB, while Ca²⁺ can boost it to 126.8 %. Furthermore, LysVPB exhibited satisfactory efficacy against strains of V. parahaemolyticus. LysVPB is an innovative phage lysin with good characteristics that are specific to certain hosts. The modular nature of LysVPB allows for efficient domain exchange with alternative lysins as antimicrobial components and fusion with antimicrobial peptides. This opens up possibilities for engineering chimeric lysins in a broader range of target hosts with high antimicrobial effectiveness and strong activity under physiological conditions.

目前，人们急需开发安全、环保的抗生素替代品来对付副溶血性弧菌。内溶酶因具有理想的作用范围和对付抗生素耐药菌的能力，被认为是很有前途的抗菌剂。虽然已经发现了许多弧菌噬菌体，但对其内溶素的研究仍处于起步阶段。本研究克隆了一种名为 LysVPB 的新型内溶酶，并在 Pichia pastoris 中进行了表达。系统进化分析表明，LysVPB 与其他已知的内溶酶几乎没有相似之处，这突显了它的独特性。同源建模确定了 LysVPB 中的一个假定钙结合位点。重组 LysVPB 的裂解活性为 64.8 U/mL，分子量约为 17 kDa。LysVPB 在 pH 值为 9.0 时具有更高的活性，而在 pH 值为 6.0-10.0 的宽泛范围内，其最大活性只有 60%。LysVPB 的催化效率在 30 °C 时达到峰值，但超过 50 °C 时则明显下降。Ba2+、Co2+ 和 Cu2+ 对 LysVPB 的活性有抑制作用，而 Ca2+ 则能将其活性提高到 126.8%。此外，LysVPB 对副溶血性弧菌菌株也表现出令人满意的功效。LysVPB 是一种创新的噬菌体溶菌酶，具有针对特定宿主的良好特性。LysVPB 的模块化特性允许与其他溶酶体进行有效的结构域交换，作为抗菌成分，并与抗菌肽融合。这就为在更广泛的目标宿主中设计具有高抗菌效力和生理条件下强活性的嵌合溶酶体提供了可能性。

{"title":"Characterization of a marine endolysin LysVPB against Vibrio parahaemolyticus","authors":"Juan Chen , Ziyun Zhao , Xiaofeng Mu , Mengxin Wang , Jun Tang , Qingqing Bi","doi":"10.1016/j.pep.2024.106608","DOIUrl":"10.1016/j.pep.2024.106608","url":null,"abstract":"<div>Currently, there is an urgent to develop safe and environmentally friendly alternatives to antibiotics for combating Vibrio parahaemolyticus. Endolysins are considered promising antibacterial agents due to their desirable range of action and ability to deal with antibiotic-resistant bacteria. While numerous Vibrio phages have been identified, the research on their endolysins is still in its infancy. In this study, a novel endolysin called LysVPB was cloned and expressed in Pichia pastoris. Phylogenetic analysis revealed that LysVPB bears little resemblance to other known endolysins, highlighting its unique nature. Homology modeling identified a putative calcium-binding site in LysVPB. The recombinant LysVPB achieved a lytic activity of 64.8 U/mL and had a molecular weight of approximately 17 kDa. LysVPB exhibited enhanced efficacy at pH 9.0, with 60 % of its maximum activity observed within the broad pH range of 6.0–10.0. The catalytic efficiency of LysVPB peaked at 30 °C but significantly declined beyond 50 °C. Ba2+, Co2+, and Cu2+ showed inhibitory effects on the activity of LysVPB, while Ca2+ can boost it to 126.8 %. Furthermore, LysVPB exhibited satisfactory efficacy against strains of V. parahaemolyticus. LysVPB is an innovative phage lysin with good characteristics that are specific to certain hosts. The modular nature of LysVPB allows for efficient domain exchange with alternative lysins as antimicrobial components and fusion with antimicrobial peptides. This opens up possibilities for engineering chimeric lysins in a broader range of target hosts with high antimicrobial effectiveness and strong activity under physiological conditions.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106608"},"PeriodicalIF":1.4,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142239835","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

Insights into the functional mechanism of the non-specific lipid transfer protein nsLTP in Kalanchoe fedtschenkoi (Lavender scallops) 对薰衣草扇贝中非特异性脂质转移蛋白 nsLTP 功能机制的认识

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-09-10 DOI: 10.1016/j.pep.2024.106607

Dafeng Liu , Wenrui Dou , Hongying Song , Huashui Deng , Zhu Tian , Rong Chen , Zhen Liu , Ziwei Jiao , Oren Akhberdi

Plant non-specific lipid transfer protein (nsLTP) is able to bind and transport lipids and essential oils, as well as engage in various physiological processes, including defense against phytopathogens. Kalanchoe fedtschenkoi (Lavender Scallops) is an attractive and versatile succulent. To investigate the functional mechanism of Kalanchoe fedtschenkoi nsLTP (Ka-nsLTP), we expressed, purified and successfully obtained monomeric Ka-nsLTP. Mutational experiments revealed that the C6A variant retained the same activity as the wild-type (WT) Ka-nsLTP. Ka-nsLTP showed weak antiphytopathogenic bacterial activity, but inhibited fungal growth. Ka-nsLTP possessed a hydrophobic cavity effectively binding lauric acid. Our results offer novel molecular insights into the functional mechanism of nsLTP, which broadens our knowledge of the biological function of nsLTP in crops and provides a useful locus for genetic improvement of plants.

植物非特异性脂质转移蛋白（nsLTP）能够结合和运输脂质和精油，并参与各种生理过程，包括抵御植物病原体。薰衣草扇贝（Kalanchoe fedtschenkoi）是一种极具吸引力且用途广泛的多肉植物。为了研究 Kalanchoe fedtschenkoi nsLTP（Ka-nsLTP）的功能机制，我们表达、纯化并成功获得了单体 Ka-nsLTP。突变实验表明，C6A变体保留了与野生型（WT）Ka-nsLTP相同的活性。Ka-nsLTP 具有较弱的抗致病细菌活性，但能抑制真菌生长。Ka-nsLTP 具有一个疏水腔，能有效结合月桂酸。我们的研究结果为 nsLTP 的功能机制提供了新的分子见解，拓宽了我们对 nsLTP 在作物中生物功能的认识，为植物遗传改良提供了一个有用的基因位点。

{"title":"Insights into the functional mechanism of the non-specific lipid transfer protein nsLTP in Kalanchoe fedtschenkoi (Lavender scallops)","authors":"Dafeng Liu , Wenrui Dou , Hongying Song , Huashui Deng , Zhu Tian , Rong Chen , Zhen Liu , Ziwei Jiao , Oren Akhberdi","doi":"10.1016/j.pep.2024.106607","DOIUrl":"10.1016/j.pep.2024.106607","url":null,"abstract":"<div>Plant non-specific lipid transfer protein (nsLTP) is able to bind and transport lipids and essential oils, as well as engage in various physiological processes, including defense against phytopathogens. Kalanchoe fedtschenkoi (Lavender Scallops) is an attractive and versatile succulent. To investigate the functional mechanism of Kalanchoe fedtschenkoi nsLTP (Ka-nsLTP), we expressed, purified and successfully obtained monomeric Ka-nsLTP. Mutational experiments revealed that the C6A variant retained the same activity as the wild-type (WT) Ka-nsLTP. Ka-nsLTP showed weak antiphytopathogenic bacterial activity, but inhibited fungal growth. Ka-nsLTP possessed a hydrophobic cavity effectively binding lauric acid. Our results offer novel molecular insights into the functional mechanism of nsLTP, which broadens our knowledge of the biological function of nsLTP in crops and provides a useful locus for genetic improvement of plants.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106607"},"PeriodicalIF":1.4,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142164833","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 and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis 来自 Rahnella aquatilis 的新型 L-isoleucine-4-dioxygenase (RaIDO) 的生物化学和结构特征。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-09-06 DOI: 10.1016/j.pep.2024.106604

Ruida Shan , Yishu Wang , Shuxin Cheng , Xia Li , Xiaohui Yang , Dengyue Sun , Piwu Li

The _L-isoleucine-4-dioxygenase converts _L-isoleucine (Ile) into(2S,3R,4S)-4-(OH)-isoleucine (4-HIL), a naturally occurring hydroxyl amino acid, which is a promising compound for drug and functional food development. Here, a novel _L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis was cloned, expressed and characterized, as one of only a few reported _L-isoleucine-4-dioxygenases. RaIDO showed high catalytic efficiency with Ile as the substrate, as well as good stability. HPLC-MS and NMR confirmed that RaIDO converts Ile into (2S,3R,4S)-4-(OH)-isoleucine. Further, structural analysis of RaIDO revealed key active site residues, including H159, D161 and H212. The RaIDO enzyme showed an optimal reaction temperature range of 30°C–45 °C, with the highest catalytic activity observed at 40 °C. Additionally, the enzyme exhibited an optimal pH of 8.0. Thus, the novel _L-isoleucine-4-dioxygenase (RaIDO) has high catalytic efficiency and good stability, making it a strong candidate for industrial applications.

L-异亮氨酸-4-二加氧酶可将 L-异亮氨酸（Ile）转化为（2S,3R,4S）-4-（OH）-异亮氨酸（4-HIL），这是一种天然存在的羟基氨基酸，是一种很有希望用于药物和功能性食品开发的化合物。本文克隆、表达并鉴定了一种新型 L-异亮氨酸-4-二加氧酶（RaIDO），它是仅有的几种报道的 L-异亮氨酸-4-二加氧酶之一。以 Ile 为底物时，RaIDO 表现出较高的催化效率和良好的稳定性。HPLC-MS 和 NMR 证实，RaIDO 能将 Ile 转化为 (2S,3R,4S)-4-(OH)-异亮氨酸。此外，RaIDO的结构分析还揭示了关键的活性位点残基，包括H159、D161和H212。RaIDO 酶的最佳反应温度范围为 30°C-45°C，在 40°C 时催化活性最高。此外，该酶的最佳 pH 值为 8.0。因此，新型 L-异亮氨酸-4-二加氧酶（RaIDO）具有催化效率高和稳定性好的特点，是工业应用的理想选择。

{"title":"Biochemical and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis","authors":"Ruida Shan , Yishu Wang , Shuxin Cheng , Xia Li , Xiaohui Yang , Dengyue Sun , Piwu Li","doi":"10.1016/j.pep.2024.106604","DOIUrl":"10.1016/j.pep.2024.106604","url":null,"abstract":"<div>The L-isoleucine-4-dioxygenase converts L-isoleucine (Ile) into(2S,3R,4S)-4-(OH)-isoleucine (4-HIL), a naturally occurring hydroxyl amino acid, which is a promising compound for drug and functional food development. Here, a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis was cloned, expressed and characterized, as one of only a few reported L-isoleucine-4-dioxygenases. RaIDO showed high catalytic efficiency with Ile as the substrate, as well as good stability. HPLC-MS and NMR confirmed that RaIDO converts Ile into (2S,3R,4S)-4-(OH)-isoleucine. Further, structural analysis of RaIDO revealed key active site residues, including H159, D161 and H212. The RaIDO enzyme showed an optimal reaction temperature range of 30°C–45 °C, with the highest catalytic activity observed at 40 °C. Additionally, the enzyme exhibited an optimal pH of 8.0. Thus, the novel L-isoleucine-4-dioxygenase (RaIDO) has high catalytic efficiency and good stability, making it a strong candidate for industrial applications.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106604"},"PeriodicalIF":1.4,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142146096","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 an effective method for purifying trypsin using a recombinant inhibitor 开发一种利用重组抑制剂纯化胰蛋白酶的有效方法。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-09-02 DOI: 10.1016/j.pep.2024.106597

Chen Li , Zhaoxia Wang , Zejie Niu , Jiao Li , Lanxin Chen , Xiaodong Cui , Fang Li

A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and K_m value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30–50 °C and pH range of 4.0–10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species.

通过在环氧氯丙烷活化的交联琼脂糖凝胶（Selfinose CL 6B）上共价固定荞麦胰蛋白酶抑制剂（BTI），制备了一种胰蛋白酶亲和材料。活化 Selfinose CL 6B 的最佳条件是 15%环氧氯丙烷和 0.8 M NaOH，温度为 40°C，时间为 2 小时；固定 BTI 的最佳 pH 值为 9.5。BTI-Sefinose CL 6B 的最大吸附容量为 2.25 毫克胰蛋白酶/（克支持物）。该材料还具有良好的重复使用性，在循环 30 次后仍能保持 90% 以上的初始吸附容量。使用 BTI-Selfinose CL 6B 通过一步亲和层析从蝗虫匀浆中获得了高纯度胰蛋白酶。以 N-苯甲酰基-DL-精氨酸-硝基苯胺为底物，测定了蝗虫胰蛋白酶的分子质量和 Km 值分别为 27 kDa 和 0.241 mM。胰蛋白酶活性的最佳温度和 pH 值分别为 55 °C 和 9.0。该酶在 30-50 °C 的温度范围和 4.0-10.0 的 pH 范围内表现出良好的稳定性。BTI-Selfinose CL 6B 在制备高纯度胰蛋白酶和从不同物种中发现更多新型胰蛋白酶方面具有潜在的应用价值。

{"title":"Development of an effective method for purifying trypsin using a recombinant inhibitor","authors":"Chen Li , Zhaoxia Wang , Zejie Niu , Jiao Li , Lanxin Chen , Xiaodong Cui , Fang Li","doi":"10.1016/j.pep.2024.106597","DOIUrl":"10.1016/j.pep.2024.106597","url":null,"abstract":"<div>A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and Km value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30–50 °C and pH range of 4.0–10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"225 ","pages":"Article 106597"},"PeriodicalIF":1.4,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133522","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