Protein expression and purification最新文献_第6页

Efficient periplasmic expression of active lysyl endopeptidase and optimizing the purification methods 活性赖氨酰内肽酶的高效外质粒表达及纯化方法的优化。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-04 DOI: 10.1016/j.pep.2024.106618

Zahra Pourani , Malihe Keramati , Samira Komijani , Majid Golkar , Reza Ahangari Cohan , Nastaran Mohseni , Vahideh Valizadeh

Recombinant production of lysyl endopeptidase (Lys-C) which is frequently used in proteomics is still challenging due to its complex structure. Herein, periplasmic expression and determining effective factors for recovery of the active enzyme were investigated. The codon-optimized Lys-C gene was cloned into pET26b (+) for periplasmic expression in E. coli Rosetta (DE3). The following parameters affecting expression level and activity of Lys-C were investigated including IPTG concentration (0.05–1 mM), cell density (OD₆₀₀: 0.45–0.8) at induction time, presence of reducing agents (glutathione or cysteine, 0–10 mM) in culture medium or periplasmic extraction buffers, and harvesting time (6 or 20 h). Lys-C was then purified by DEAE and Ni-NTA chromatography methods. The highest expression level was obtained at 0.05 mM IPTG (5.49 %), also 8 mM cysteine, induction at OD_600: 0.45 and 6 h incubation increased enzyme activity to 23.5 %, 13.3 %, and 76.4 %, respectively. The enzyme activity of Lys-C in the presence of 4 mM glutathione and extraction buffers containing 2 mM 2-mercaptoethanol (2 ME) was 81.6 % higher than the condition without reducing agents. Also, 8 mM cysteine in the culture medium and 2 mM 2 ME in extraction increased the activity up to 29.7 %. Moreover, optimization of purification process enhanced the enzyme activity from 0.217 mU to 1.76 mU. Statistical analysis showed the examined parameters significantly affected enzyme activity (p < 0.05). The presence of the reducing agents in the culture medium and extraction buffers presumably improves the Lys-C folding and increases the enzyme activity.

蛋白质组学中常用的赖氨酰内肽酶（Lys-C）结构复杂，因此其重组生产仍具有挑战性。本文研究了外质粒表达和确定回收活性酶的有效因素。将经过密码子优化的 Lys-C 基因克隆到 pET26b (+) 中，在大肠杆菌 Rosetta (DE3) 中进行质粒表达。研究了影响 Lys-C 表达水平和活性的参数，包括 IPTG 浓度（0.05-1 mM）、诱导时的细胞密度（OD600：0.45-0.8）、培养基或质外提取缓冲液中还原剂（谷胱甘肽或半胱氨酸，0-10 mM）的存在以及收获时间（6 或 20 小时）。然后用 DEAE 和 Ni-NTA 色谱法纯化 Lys-C。在 0.05 mM IPTG（5.49%）和 8 mM 半胱氨酸条件下，表达水平最高；在 OD600：0.45 和 6 小时孵育条件下，酶活性分别提高到 23.5%、13.3% 和 76.4%。在 4 mM 谷胱甘肽和含有 2 mM 2-巯基乙醇（2ME）的提取缓冲液存在下，Lys-C 的酶活性比不使用还原剂的条件下高 81.6%。此外，在培养基中加入 8 mM 半胱氨酸和在提取过程中加入 2 mM 2ME 可使活性提高 29.7%。此外，优化纯化过程可将酶活性从 0.217 mU 提高到 1.76 mU。统计分析显示，所研究的参数对酶活性有显著影响（p

{"title":"Efficient periplasmic expression of active lysyl endopeptidase and optimizing the purification methods","authors":"Zahra Pourani , Malihe Keramati , Samira Komijani , Majid Golkar , Reza Ahangari Cohan , Nastaran Mohseni , Vahideh Valizadeh","doi":"10.1016/j.pep.2024.106618","DOIUrl":"10.1016/j.pep.2024.106618","url":null,"abstract":"<div><div>Recombinant production of lysyl endopeptidase (Lys-C) which is frequently used in proteomics is still challenging due to its complex structure. Herein, periplasmic expression and determining effective factors for recovery of the active enzyme were investigated. The codon-optimized Lys-C gene was cloned into pET26b (+) for periplasmic expression in <em>E. coli</em> Rosetta (DE3). The following parameters affecting expression level and activity of Lys-C were investigated including IPTG concentration (0.05–1 mM), cell density (OD<sub>600</sub>: 0.45–0.8) at induction time, presence of reducing agents (glutathione or cysteine, 0–10 mM) in culture medium or periplasmic extraction buffers, and harvesting time (6 or 20 h). Lys-C was then purified by DEAE and Ni-NTA chromatography methods. The highest expression level was obtained at 0.05 mM IPTG (5.49 %), also 8 mM cysteine, induction at OD<sub>600:</sub> 0.45 and 6 h incubation increased enzyme activity to 23.5 %, 13.3 %, and 76.4 %, respectively. The enzyme activity of Lys-C in the presence of 4 mM glutathione and extraction buffers containing 2 mM 2-mercaptoethanol (2 ME) was 81.6 % higher than the condition without reducing agents. Also, 8 mM cysteine in the culture medium and 2 mM 2 ME in extraction increased the activity up to 29.7 %. Moreover, optimization of purification process enhanced the enzyme activity from 0.217 mU to 1.76 mU. Statistical analysis showed the examined parameters significantly affected enzyme activity (p < 0.05). The presence of the reducing agents in the culture medium and extraction buffers presumably improves the Lys-C folding and increases the enzyme activity.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106618"},"PeriodicalIF":1.4,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142591338","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

Human parvovirus B19 virus-like particle formation in Nicotiana benthamiana 人副病毒 B19 病毒样颗粒在烟草中的形成。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-10-31 DOI: 10.1016/j.pep.2024.106616

Sakika Kimura , Jiahui Ong , Atsushi Kasai , Shinji Akada , Hirotaka Ebina , Michiko Sasabe , Eiji Morita

There has been a surge in the interest to utilize plants as hosts for producing vaccine antigens. In this study, we demonstrated the successful expression of the human parvovirus B19 (B19V) capsid protein (VP2) in Nicotiana benthamiana cells. The B19V VP1 and VP2 genes were cloned under the control of estrogen-inducible promoters and transiently expressed in N. benthamiana leaves using the agroinfiltration method. The addition of estrogen significantly boosted the expression of VP2. Furthermore, codon optimization of the VP2 sequence resulted in over a 30-fold increase in its expression compared with that of the wild-type. Analysis of negatively stained samples by sucrose density gradient ultracentrifugation and electron microscopy revealed that the expressed VP2 proteins formed spherical particles with diameters of approximately 20 nm. Immunostaining analysis of protoplasts derived from VP2-expressing N. benthamiana leaves indicated that VP2 signals were predominantly localized in the cytoplasm. These findings strongly suggested that B19V VP2 assembles and formed virus-like particles (VLPs) within the cytoplasm of N. benthamiana cells, presenting a promising method for producing B19V VLPs in plant systems.

利用植物作为宿主生产疫苗抗原的兴趣日益高涨。在这项研究中，我们证明了人副病毒 B19（B19V）荚膜蛋白（VP2）在烟草根细胞中的成功表达。在雌激素诱导启动子的控制下克隆了 B19V VP1 和 VP2 基因，并采用农渗法在烟草叶片中进行了瞬时表达。加入雌激素可明显促进 VP2 的表达。此外，对 VP2 序列进行密码子优化后，其表达量比野生型提高了 30 多倍。通过蔗糖密度梯度超速离心和电子显微镜对负染样品进行分析，发现表达的 VP2 蛋白形成了直径约为 20 纳米的球形颗粒。对来自表达 VP2 的 N. benthamiana 叶片的原生质体进行的免疫染色分析表明，VP2 信号主要定位于细胞质中。这些发现有力地表明，B19V VP2 在 N. benthamiana 细胞的细胞质中组装并形成病毒样颗粒（VLPs），为在植物系统中生产 B19V VLPs 提供了一种可行的方法。

{"title":"Human parvovirus B19 virus-like particle formation in Nicotiana benthamiana","authors":"Sakika Kimura , Jiahui Ong , Atsushi Kasai , Shinji Akada , Hirotaka Ebina , Michiko Sasabe , Eiji Morita","doi":"10.1016/j.pep.2024.106616","DOIUrl":"10.1016/j.pep.2024.106616","url":null,"abstract":"<div><div>There has been a surge in the interest to utilize plants as hosts for producing vaccine antigens. In this study, we demonstrated the successful expression of the human parvovirus B19 (B19V) capsid protein (VP2) in <em>Nicotiana benthamiana</em> cells. The B19V VP1 and VP2 genes were cloned under the control of estrogen-inducible promoters and transiently expressed in <em>N. benthamiana</em> leaves using the agroinfiltration method. The addition of estrogen significantly boosted the expression of VP2. Furthermore, codon optimization of the VP2 sequence resulted in over a 30-fold increase in its expression compared with that of the wild-type. Analysis of negatively stained samples by sucrose density gradient ultracentrifugation and electron microscopy revealed that the expressed VP2 proteins formed spherical particles with diameters of approximately 20 nm. Immunostaining analysis of protoplasts derived from VP2-expressing <em>N. benthamiana</em> leaves indicated that VP2 signals were predominantly localized in the cytoplasm. These findings strongly suggested that B19V VP2 assembles and formed virus-like particles (VLPs) within the cytoplasm of <em>N. benthamiana</em> cells, presenting a promising method for producing B19V VLPs in plant systems.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106616"},"PeriodicalIF":1.4,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564768","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

Detection and optimization of microbial expression systems for extracellular production and purification of Ca2+-responsive phase-changing annexin fusions 检测和优化用于细胞外生产和纯化 Ca2+ 反应相变附件蛋白融合体的微生物表达系统。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-10-30 DOI: 10.1016/j.pep.2024.106617

Jinjing Li, Baokang Wu, Yiting Ji, Shuncheng Zhang, Yuanyuan Ge, Jun Fan

Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca²⁺ and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost.

在此之前，我们发现人类附件素 A1 是一种纯化标签，可通过更温和的钙响应沉淀进行无柱纯化。在这项工作中，我们利用附件蛋白 A1 标记的绿色荧光蛋白构建体检测了大肠杆菌、枯草芽孢杆菌和 Pichia pastoris 的胞外产物，发现无领导融合蛋白在大肠杆菌中通过 Triton X-100 等添加剂进行胞外转运。对大肠杆菌胞外表达系统中的共表达酶、培养成分和诱导条件进行了优化。在 28 ℃ 诱导 60 小时后，加入 0.2% Triton X-100，与蜡样芽孢杆菌的磷脂酶 C 共表达，大肠杆菌的附件蛋白 A1 标记绿色荧光蛋白和 5-氨基乙酰脱水酶被过量表达，并通过 20 mM Ca2+ 沉淀和 25 mM EDTA 再溶解从溶菌酶肉汤中纯化，蛋白质纯度和回收率均可接受。硅胶结合肽与附件蛋白 A1 标记的荧光蛋白融合，进行连续的亲和沉淀和纯化。在特异性蛋白酶的作用下，通过树脂裂解释放的无标签蛋白的纯度高于裂解游离融合蛋白的纯度。串联标签适用于培养物中少量或大量其他融合蛋白的两步纯化，并能以低成本回收不含标签的蛋白。

{"title":"Detection and optimization of microbial expression systems for extracellular production and purification of Ca2+-responsive phase-changing annexin fusions","authors":"Jinjing Li, Baokang Wu, Yiting Ji, Shuncheng Zhang, Yuanyuan Ge, Jun Fan","doi":"10.1016/j.pep.2024.106617","DOIUrl":"10.1016/j.pep.2024.106617","url":null,"abstract":"<div><div>Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in <em>Escherichia coli</em>, <em>Bacillus subtilis</em>, and <em>Pichia pastoris</em>, and identified that the leaderless fusion protein was transported extracellularly in <em>E. coli</em> with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in <em>E. coli</em> extracellular expression systems were optimized. With coexpression of phospholipase C from <em>Bacillus cereus</em> and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from <em>E. coli</em> were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca<sup>2+</sup> and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106617"},"PeriodicalIF":1.4,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564752","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 the chimera proteins of Nav1.7 and NavAb in Escherichia coli Nav1.7 和 NavAb 嵌合体蛋白在大肠杆菌中的功能表达。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-10-30 DOI: 10.1016/j.pep.2024.106615

Tomohiro Yamaguchi, Toshiaki Okada, Tadashi Kimura

Na_v1.7 is a eukaryotic voltage-dependent Na channel (Na_v) family membrane protein and has four channel domains and four voltage sensor domains (VSD-I–IV). It is involved in pain perception, and VSDs that differ significantly by Na_v subtype are targeted in the development of Na_v1.7-specific inhibitors. This is expected to result in neuropathic pain treatments with fewer side effects. We previously reported on intra-periplasm secretion and selection (PERISS), a peptide drug discovery system that targets membrane proteins by co-expressing a peptide library and a target membrane protein. For PERISS screening of VSD-specific new Na_v1.7 inhibitors, the chimera protein (Na_vAb/1.7VSD) of Na_v from prokaryotic Arcobacter butzleri (Na_vAb), in which extracellular loops of VSD were replaced with homologous loops from Na_v1.7, serves as an effective model. This is because Na_vAb harbors only one VSD and the biological activity of Na_vAb/1.7VSD was previously confirmed. To date, Na_vAb/1.7VSD has only been found to be expressed in insect cells. In this study, we report on the expression and channel activity of Na_vAb/1.7VSD-II in Escherichia coli (E. coli). The expression of this protein in the inner membrane of E. coli was confirmed by western blotting. Channel activity was assessed by measuring the channel currents of the purified recombinant proteins and inhibition using a Na_v1.7-specific peptide inhibitor. The results indicate that Na_vAb/1.7VSD-II was functionally expressed in E. coli, providing empirical support for the discovery of new VSD-specific Na_v1.7 inhibitors using the PERISS screening method.

Nav1.7 是真核生物电压依赖性 Na 通道（Nav）家族的膜蛋白，具有四个通道结构域和四个电压传感器结构域（VSD-I-IV）。它参与痛觉感知，在开发 Nav1.7 特异性抑制剂时，Nav1.7 亚型中差异显著的 VSD 是目标。这有望减少神经病理性疼痛治疗的副作用。我们以前曾报道过质内分泌和选择（PERISS），这是一种多肽药物发现系统，通过共同表达多肽库和目标膜蛋白来靶向膜蛋白。为了通过 PERISS 筛选 VSD 特异性的 Nav1.7 抑制剂，原核生物 Arcobacter butzleri 的 Nav 嵌合蛋白（NavAb/1.7VSD）成为了一个有效的模型。这是因为 NavAb 只含有一个 VSD，而且 NavAb/1.7VSD 的生物活性已被证实。迄今为止，NavAb/1.7VSD 只被发现在昆虫细胞中表达。在这项研究中，我们报告了 NavAb/1.7VSD-II 在大肠杆菌（E. coli）中的表达和通道活性。该蛋白在大肠杆菌内膜中的表达是通过 Western 印迹法证实的。通过测量纯化重组蛋白的通道电流和使用 Nav1.7 特异性肽抑制剂的抑制作用，对通道活性进行了评估。结果表明，NavAb/1.7VSD-II 在大肠杆菌中得到了功能表达，为使用 PERISS 筛选方法发现新的 VSD 特异性 Nav1.7 抑制剂提供了经验支持。

{"title":"Functional expression of the chimera proteins of Nav1.7 and NavAb in Escherichia coli","authors":"Tomohiro Yamaguchi, Toshiaki Okada, Tadashi Kimura","doi":"10.1016/j.pep.2024.106615","DOIUrl":"10.1016/j.pep.2024.106615","url":null,"abstract":"<div><div>Na<sub>v</sub>1.7 is a eukaryotic voltage-dependent Na channel (Na<sub>v</sub>) family membrane protein and has four channel domains and four voltage sensor domains (VSD-I–IV). It is involved in pain perception, and VSDs that differ significantly by Na<sub>v</sub> subtype are targeted in the development of Na<sub>v</sub>1.7-specific inhibitors. This is expected to result in neuropathic pain treatments with fewer side effects. We previously reported on intra-periplasm secretion and selection (PERISS), a peptide drug discovery system that targets membrane proteins by co-expressing a peptide library and a target membrane protein. For PERISS screening of VSD-specific new Na<sub>v</sub>1.7 inhibitors, the chimera protein (Na<sub>v</sub>Ab/1.7VSD) of Na<sub>v</sub> from prokaryotic <em>Arcobacter butzleri</em> (Na<sub>v</sub>Ab), in which extracellular loops of VSD were replaced with homologous loops from Na<sub>v</sub>1.7, serves as an effective model. This is because Na<sub>v</sub>Ab harbors only one VSD and the biological activity of Na<sub>v</sub>Ab/1.7VSD was previously confirmed. To date, Na<sub>v</sub>Ab/1.7VSD has only been found to be expressed in insect cells. In this study, we report on the expression and channel activity of Na<sub>v</sub>Ab/1.7VSD-II in <em>Escherichia coli</em> (<em>E. coli</em>). The expression of this protein in the inner membrane of <em>E. coli</em> was confirmed by western blotting. Channel activity was assessed by measuring the channel currents of the purified recombinant proteins and inhibition using a Na<sub>v</sub>1.7-specific peptide inhibitor. The results indicate that Na<sub>v</sub>Ab/1.7VSD-II was functionally expressed in <em>E. coli</em>, providing empirical support for the discovery of new VSD-specific Na<sub>v</sub>1.7 inhibitors using the PERISS screening method.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106615"},"PeriodicalIF":1.4,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564756","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 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 <em>Escherichia coli</em> (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 <sub>DC</sub>Survivin-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 <em>Aspergillus awamori</em> exo-inulinase 1 are compared for <em>A. awamori</em> and <em>Penicillium verruculosum</em> expression hosts. The tertiary structure contains seven sites of <em>N</em>-glycosylation, with two of them located near the active center. If expressed in <em>P. verruculosum</em>, the enzyme was four times less glycosylated and two times more active toward sucrose, raffinose, and stachyose due to an increase in <em>k</em><sub><em>cat</em></sub>. 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 <em>N</em>-linked glycans. Molecular dynamics simulations showed an increase in the fluctuation of the active center with an increase in the length of <em>N</em>-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 Ca<sup>2+</sup>-calmodulin (CaM) activated enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. Our prior research established that <em>in vivo</em> phosphorylation of AtGAD1 (AT5G17330) occurs at multiple N-terminal serine residues following Pi resupply to Pi-starved cell cultures of the model plant <em>Arabidopsis thaliana</em>. The aim of the current investigation was to purify recombinant AtGAD1 (rAtGAD1) following its expression in <em>Escherichia coli</em> to facilitate studies of the impact of phosphorylation on its kinetic properties. However, <em>in vitro</em> 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 <em>E. coli</em> cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca<sup>2+</sup>/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 Ca<sup>2+</sup>/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 Ca<sup>2+</sup>/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 <em>E. coli</em> 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 <em>E. coli</em> system. Moreover, the indirect ELISA assay developed using Bac-E2 protein exhibited superior specificity, sensitivity, and accuracy in comparison to the <em>E. coli</em>-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 <em>Lactobacillus plantarum</em> PTCC 1745 on the biofilm formations of <em>A</em>. <em>baumannii</em> isolates. Bacteriocin derived from <em>L</em>. <em>plantarum</em> 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 <em>bap</em> gene expression in bacteriocin-treated cells. According to SDS-PAGE, bacteriocin from <em>L</em>. <em>plantarum</em> 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 <em>bap</em> gene among the MDR <em>A</em>. <em>baumannii</em> isolates decreased by 52 % compared to the untreated control. This study demonstrated that bacteriocin derived from <em>L</em>. <em>plantarum</em> PTCC 1745 had antibacterial and antibiofilm activity against MDR <em>A</em>. <em>baumannii</em> 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