Protein expression and purification最新文献_第5页

Expression of the Fusarium graminearum galactose oxidase GaoA in Saccharomyces cerevisiae 谷草镰刀菌半乳糖氧化酶gaa在酿酒酵母中的表达。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-29 DOI: 10.1016/j.pep.2024.106637

Lucas Yudai Nozaki, Nathalia Rodrigues Bulka, Karina Lima dos Reis, Damaris Batistão Martim, Fausto Fernandes de Castro, Ione Parra Barbosa-Tessmann

Galactose oxidase, produced by fungi of the genus Fusarium, is an enzyme of great biotechnological importance. The gaoA gene has been recombinantly expressed in several hosts but has yet to be in Saccharomyces cerevisiae. This work aimed to express the Fusarium graminearum GaoA enzyme in S. cerevisiae. The full-length and the truncated F. graminearum gaoA gene were subcloned into a yeast expression vector. The GaoA enzyme expression level in S. cerevisiae was higher when the truncated gene, which codes for the mature form of the enzyme, was used. After purification of the expressed enzyme on a Sepharose® 6B column, the obtained yield of the pure and active enzyme was 16.7 mg/L. The purified protein showed a K_M of 9.8 mM, lower than that of the wild-type enzyme, and a k_cat/K_M of 2.9 × 10⁷ M⁻¹s⁻¹, higher than that of the wild-type enzyme. The expressed recombinant protein used several common substrates for galactose oxidase, such as galactose, raffinose, and 1,3-dihydroxyacetone dimer. In addition, it had increased activity on guar gum, lactose, and Arabic gum compared with the wild-type enzyme. The obtained enzyme's characteristics are compatible with the galactose oxidase biotechnological applications.

半乳糖氧化酶是由镰刀菌属真菌产生的一种具有重要生物技术意义的酶。gaoA基因已在几种宿主中重组表达，但尚未在酿酒酵母中表达。本研究的目的是在酿酒酵母中表达谷草镰刀菌GaoA酶。将小麦赤霉病菌gaoA基因全长和截短片段亚克隆到酵母表达载体上。当截断的基因编码成熟形式的GaoA酶时，酿酒酵母中GaoA酶的表达量更高。表达酶经Sepharose 6B柱纯化后，得到纯活性酶的产率为16.7 mg/L。纯化后的蛋白KM为9.8 mM，低于野生型酶，kcat/KM为2.9 × 107 M-1s-1，高于野生型酶。所表达的重组蛋白使用了几种常见的半乳糖氧化酶底物，如半乳糖、棉子糖和1,3-二羟基丙酮二聚体。此外，与野生型酶相比，它对瓜尔胶、乳糖和阿拉伯胶的活性增加。所得酶的特性与半乳糖氧化酶的生物技术应用相适应。

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引用次数: 0

The combined effect of the gene copy number and chaperone overexpression on the recombinant bovine chymosin production in Pichia pastoris, with mutant ADH2 promoter 基因拷贝数和伴侣蛋白过表达对ADH2启动子突变的毕赤酵母重组牛凝乳酶产生的联合影响。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-29 DOI: 10.1016/j.pep.2024.106636

Fatma Ersöz , Mehmet İnan

Chymosin is an enzyme used to coagulate milk, in the cheese industry. This study aimed to increase recombinant production of the chymosin in Pichia pastoris by determining the optimum copy number and overproduction of a Protein Disulfide Isomerase (PpPDI) chaperon protein. Bos taurus chymosin was expressed under the control of a mutant ADH2 promoter. The clones containing 1–4 gene copy numbers of the chymosin were constructed using the in vitro cloning method, and the effect of chaperone protein on chymosin secretion was investigated.

The enzyme production levels are 4, 6.3, 4.5, and 3 IMCU/mL for 1, 2, 3, and 4-copy clones. The secreted chymosin levels increased up to two copies, and increasing the number of copies decreased the secretion level. Therefore, PpPDI was over-expressed in the clones regulated with the ADH2 promoter. The over-expression of PDI gene increased chymosin secretion in clones compared to the counterpart host. However, the highest chymosin level was obtained with C2 (2-copy chymosin containing clone; 6.3 IMCU/mL) and C2P2 (2-copy chymosin/2-copy PDI containing clone; 8.2 IMCU/mL).

The maximum production was 39 IMCU/mL with the clone C2P2 in the fermenter scale production. The enzyme activity increased approximately 2-fold by adding two copies of the chaperone protein. The combined effect of gene copy number and chaperone overexpression on chymosin production was investigated. Two copies of the chymosin and PpPDI genes were the optimum among the tested clones.

凝乳酶是一种在奶酪工业中用于凝固牛奶的酶。本研究旨在通过确定蛋白二硫异构酶（PpPDI）伴侣蛋白的最佳拷贝数和过量生产来增加毕赤酵母中凝乳酶的重组生产。牛凝乳酶在ADH2启动子突变体的控制下表达。采用体外克隆的方法构建了含有1 ~ 4个基因拷贝数的乳糜蛋白酶克隆，并研究了蛋白伴侣对乳糜蛋白酶分泌的影响。1、2、3和4拷贝克隆的酶产量分别为4、6.3、4.5和3 IMCU/mL。分泌的凝乳酶增加到2个拷贝，拷贝数的增加使分泌水平降低。因此，PpPDI在ADH2启动子调控的克隆中过表达。与对应宿主相比，PDI基因的过表达增加了克隆的凝乳酶分泌。然而，C2（2拷贝）含凝乳酶克隆的凝乳酶水平最高；6.3 IMCU/mL)和C2P2(2拷贝凝乳酶/2拷贝含PDI的克隆；8.2 IMCU /毫升)。克隆C2P2在发酵罐规模生产中最高产量为39 IMCU/mL。通过添加两个伴侣蛋白的拷贝，酶活性增加了大约2倍。研究了基因拷贝数和伴侣蛋白过表达对凝乳酶产生的共同影响。其中凝乳酶基因和PpPDI基因的两个拷贝最优。

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引用次数: 0

Efficient purification and excitation energy transfer characterization of phycoerythrin 545 from Rhodomonas sp. Rhodomonas藻红蛋白545的高效纯化及激发能转移特性研究

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-26 DOI: 10.1016/j.pep.2024.106634

Yang Pu , Shuo Dong , Jiayu Wang , Min Li , Kai Dong , Wenjun Li , Zhihong Tang

Cryptomonad phycoerythrin 545 (PE545) is an important type of phycobiliprotein in basic research and technological innovations. Herein, we report a minimalistic hydrophobic chromatography method for its purification. High purity was achieved, with a purity ratio (A₅₄₅/A₂₈₀) of 13.66 and a recovery ratio of 78.63 %. Following SDS-PAGE, Coomassie Brilliant Blue staining revealed three bands at 9 kDa, 10 kDa, and 20 kDa, corresponding to α₁, α₂ and β subunits. Multiple spectral characteristics were analyzed to ensure that optical activity was consistent with that of the natural protein. Absorption and fluorescence spectroscopies of purified PE545 displayed a strong absorption peak at 545 nm, a shoulder peak at 564 nm, and a fluorescence emission peak at 587 nm, which confirmed unchanged energy transfer properties. Furthermore, the structural and functional integrity, especially the existence of strongly coupled central chromophore pairs with excitation delocalization, was verified by circular dichroism and ultrafast absorption spectroscopy. From the studies of ultrafast absorption spectroscopy of excitation energy transfer (EET) of PE545, four decay components with lifetimes at 0.5 ps, 2.2 ps, 63 ps, and 3000 ps were obtained. In addition, the dynamics of these components confirmed the EET pathways from the central PEB chromophore pairs to the peripheral pigments and localized in the lowest state. Our work will be of considerable value for both fundamental research and applications of PE545.

隐单胞菌藻红蛋白545 （PE545）是基础研究和技术创新中重要的一类藻胆蛋白。在此，我们报告了一种极简疏水色谱法纯化其。A545/A280的纯度比为13.66，回收率为78.63%。SDS-PAGE后，考马斯亮蓝染色在9 kDa、10 kDa和20 kDa处显示三条条带，分别对应α1、α2和β亚基。分析了多种光谱特征，以确保光学活性与天然蛋白质一致。纯化后的PE545在545 nm处有一个强吸收峰，在564 nm处有一个肩峰，在587 nm处有一个荧光发射峰，证实了PE545的能量转移特性没有变化。此外，通过圆二色性和超快吸收光谱验证了结构和功能的完整性，特别是存在具有激发离域的强耦合中心发色团对。通过对PE545的激发能转移（EET）超快吸收光谱的研究，得到了4个寿命分别为0.5 ps、2.2 ps、63 ps和3000 ps的衰变组分。此外，这些组分的动力学证实了EET从PEB中心发色团对到周围色素的途径，并定位在最低状态。我们的工作对于PE545的基础研究和应用都具有重要的价值。

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引用次数: 0

Isolation and crystallization of copper resistance protein B (CopB) from Acinetobacter baumannii 鲍曼不动杆菌铜抗性蛋白B （CopB）的分离与结晶

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-26 DOI: 10.1016/j.pep.2024.106635

Niloofar Nayeri , Kamil Górecki , Karin Lindkvist-Petersson , Pontus Gourdon , Ping Li

Acinetobacter baumannii (A. baumannii) is an opportunistic, Gram-negative human pathogen, which is predominantly found in hospital patients. Its antimicrobial resistance is escalating, leading to less efficient treatments, and an increasing interest in identifying new therapeutic drugs. Metals as antimicrobials are vital in healthcare and agriculture, and copper-containing surfaces are known to reduce microbial counts, also in clinical settings. Indeed, copper (Cu) is an essential element required for survival in all organisms from bacteria to humans, but nevertheless elevated levels are highly toxic for cells. Through different regulatory mechanisms, cells maintain Cu homeostasis, and ion channels and transporters are critical in this process. Precise understanding of such ion transport requires insight into the protein structures of the involved proteins, which will also provide information important for applied sciences. Considering the medical significance of A. baumannii and the possibility to exploit Cu to handle such infections, channels and transporters represent appealing targets. Here we approached the putative outer membrane CopB (Copper resistance protein B) from A. baumannii that is postulated to conduct Cu, with characterization of its structure and function as well as to enable rational drug-design. To this end, we demonstrate in this work procedures to produce purified sample and to recover diffracting protein crystals of CopB. The protein was overproduced in E. coli and membrane extracted in a range of detergents. The solubilized protein was subjected to crystallization, which yielded hits that scatter X-rays to low resolution. Our findings have the potential to pave the way for subsequent drug discovery.

鲍曼不动杆菌（鲍曼不动杆菌）是一种机会性革兰氏阴性人类病原体，主要见于医院患者。它的抗菌素耐药性正在升级，导致治疗效率降低，人们对确定新的治疗药物的兴趣越来越大。金属作为抗菌剂在医疗保健和农业中至关重要，并且已知含铜表面可以减少微生物数量，在临床环境中也是如此。事实上，从细菌到人类，铜是所有生物生存所必需的基本元素，但铜含量升高对细胞有很大的毒性。细胞通过不同的调控机制维持Cu稳态，离子通道和转运体在这一过程中起着至关重要的作用。对这种离子传输的精确理解需要深入了解相关蛋白质的蛋白质结构，这也将为应用科学提供重要的信息。考虑到鲍曼不动杆菌的医学意义以及利用铜来处理此类感染的可能性，通道和转运体是有吸引力的目标。在这里，我们研究了鲍曼不动杆菌的外膜CopB（铜抗性蛋白B），它被认为可以传导铜，并对其结构和功能进行了表征，从而使合理的药物设计成为可能。为此，我们在本工作中演示了制备纯化样品和回收CopB衍射蛋白晶体的程序。这种蛋白质在大肠杆菌和一系列洗涤剂中提取的膜中过量产生。溶解后的蛋白质经过结晶处理，产生的撞击使x射线散射到低分辨率。我们的发现有可能为后续的药物发现铺平道路。

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引用次数: 0

Expression and purification of the intact bacterial ergothioneine transporter EgtU 完整细菌麦角硫因转运体 EgtU 的表达和纯化。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-20 DOI: 10.1016/j.pep.2024.106633

Katherine A. Edmonds, Karla Diaz-Rodriguez, David P. Giedroc

The bacterial ATP-binding cassette (ABC) transporter EgtU is responsible for uptake of the cellular antioxidant ergothioneine in Streptococcus pneumoniae, and it has homologs in a surprisingly diverse range of microbial pathogens. Crystal structures have been reported for the solute binding domain of EgtU, but many details of the structure and function of the intact heterotetrameric transporter remain to be elucidated. In this study, we have expressed S. pneumoniae EgtU and purified it from E. coli BL21 (DE3) with high purity and homogeneity. Our preliminary data establish ergothioneine binding and ATP hydrolysis by the full-length transporter solubilized in DDM micelles. Our workflow allows for isolation of suitable quantities of EgtU for ongoing structural studies and detailed biophysical characterization.

细菌 ATP 结合盒（ABC）转运体 EgtU 负责摄取肺炎链球菌细胞中的抗氧化剂麦角硫因，它在多种微生物病原体中都有同源物，其种类之多令人惊讶。EgtU 的溶质结合结构域的晶体结构已有报道，但完整的异构四聚体转运体的结构和功能的许多细节仍有待阐明。在本研究中，我们表达了肺炎双球菌的 EgtU，并从大肠杆菌 BL21 (DE3) 中纯化出了高纯度和高均匀度的 EgtU。我们的初步数据证实了在 DDM 胶束中溶解的全长转运体与麦角硫因的结合和 ATP 的水解。我们的工作流程可以分离出适当数量的 EgtU，用于正在进行的结构研究和详细的生物物理表征。

引用次数: 0

Recombinant human FOXJ1 protein binds DNA, forms higher-order oligomers, has gel-shifting domains and contains intrinsically disordered regions 重组人 FOXJ1 蛋白可结合 DNA、形成高阶寡聚体、具有凝胶转移结构域并含有内在紊乱区。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-15 DOI: 10.1016/j.pep.2024.106622

Shashank Arora, Pawan Nagarkar, Jacinta S. D'Souza

Forkhead box protein J1 (FOXJ1) is the key transcriptional regulator during the conversion of mammalian primary cilium with a 9 + 0 architecture to the motile (9 + 2) one. The nucleotide sequences of the full-length and DNA-binding domain (DBD) of the open reading frame (ORF) were isolated and expressed into E. coli as 6xHis-tagged proteins. Upon induction, the DBD formed inclusion bodies that solubilized with 8 M urea. No induction of 6xHis-FOXJ1 protein was seen despite sub-cloning into several expression vectors and E. coli host strains. To improve induction and solubility, the 6xHis tag was substituted with Glutathione S-transferase (GST), and weak induction was seen in E. coli BL21(DE3). The GST-FOXJ1 showed anomalous migration on denaturing gel electrophoresis (AM-DRE), migrating at approximately 83 kDa instead of its calculated molecular weight (Mr) of 72.4 kDa. It was also unstable and led to degradation products. The 6xHis tag was substituted with Glutathione S-transferase (GST) to improve induction and solubility. Codon-optimization improved the induction, but the protein still showed AM-DRE and instability. It seemed that the recombinant protein was either toxic or posed a metabolic burden to the E. coli cells or, once produced was prone to degradation due mainly to the lack of post-translational modification (PTM). This process is required for some eukaryotic proteins after they are manufactured in the ribosomal factory. Both the purified recombinant proteins exhibited cysteine-induced oligomerization via the formation of disulphide bridges since this was reduced using dithiothreitol (DTT). Both were equally functional as these individually bound to an oligonucleotide, a consensus DNA-binding sequence for FOX proteins. Further, the recombinant polypeptides corresponding to the C-terminus and N-terminus show anomalies indicating that the highly acidic residues (known as polyacidic gel-shifting domains) in these polypeptides contribute to the AM-DRE. We demonstrate for the first time that the recombinant HsFOXJ1 and its DBD bind to DNA, its polyacidic gel-shifting domains are the reason for the AM-DRE, is unstable leading to degradation products, exhibits cysteine-induced oligomerization and harbours intrinsically disordered regions.

叉头盒蛋白 J1（FOXJ1）是哺乳动物初级纤毛从 9+0 结构向运动（9+2）结构转化过程中的关键转录调节因子。我们分离了开放阅读框（ORF）的全长和DNA结合域（DBD）的核苷酸序列，并将其表达到大肠杆菌中，成为6xHis标记的蛋白质。诱导后，DBD 形成包涵体，并用 8 M 尿素溶解。尽管将 6xHis-FOXJ1 蛋白亚克隆到多种表达载体和大肠杆菌宿主菌株中，但仍未发现 6xHis-FOXJ1 蛋白被诱导。为了提高诱导性和可溶性，用谷胱甘肽 S-转移酶（GST）取代了 6xHis 标记，在大肠杆菌 BL21(DE3) 中发现了微弱的诱导作用。GST-FOXJ1 在变性凝胶电泳（AM-DRE）中显示出异常迁移，迁移分子量约为 83 kDa，而不是其计算分子量（Mr）72.4 kDa。它还不稳定，会产生降解产物。用谷胱甘肽 S-转移酶（GST）取代 6xHis 标记以提高诱导性和溶解性，代码优化提高了诱导性，但蛋白质仍显示 AM-DRE，且不稳定。重组蛋白似乎对大肠杆菌细胞有毒性或造成代谢负担，或者一旦产生就容易降解，主要原因是缺乏翻译后修饰（PTM）。一些真核蛋白质在核糖体工厂制造后需要进行这一过程。两种纯化的重组蛋白都表现出半胱氨酸通过形成二硫键诱导的寡聚化，因为使用二硫苏糖醇（DTT）可将其还原。这两种蛋白的功能相同，都能单独与寡核苷酸结合，而寡核苷酸是 FOX 蛋白的一种共识 DNA 结合序列。此外，与 C 端和 N 端相对应的重组多肽显示出异常，表明这些多肽中的高酸性残基（称为多酸性凝胶转移结构域）有助于 AM-DRE。我们首次证明，重组的 HsFOXJ1 及其 DBD 可与 DNA 结合，其多酸性凝胶移动结构域是产生 AM-DRE 的原因，它不稳定，会产生降解产物，表现出半胱氨酸诱导的寡聚化，并含有内在无序区。

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引用次数: 0

Thermostable phenylacetic acid degradation protein TtPaaI from Thermus thermophilus as a scaffold for tetravalent display of proteins 嗜热菌的热稳定性苯乙酸降解蛋白 TtPaaI 作为蛋白质四价展示的支架。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-12 DOI: 10.1016/j.pep.2024.106623

Aleksandra Chorążewska, Darragh Regan, Marta Kalka, Krzysztof Ciura, Natalia Porębska, Łukasz Opaliński

Numerous proteins in nature strictly require oligomerization for their full activity. Moreover, the function of natural and artificial proteins can me adjusted by altering their oligomeric state, leading to development of biotechnologically-relevant biomacromolecules. Oligomerization scaffolds from natural sources and designed de novo enable shuffling the oligomeric state and valency of biomacromolecules. In this report we probed the scaffolding potential of the thermostable phenylacetic acid degradation protein acyl-CoA from Thermus thermophilus (TtPaaI). We designed and successfully produced the fusion protein between TtPaaI (scaffold) and galectin-7, a multifunctional lectin implicated in human diseases (ligand) and demonstrated that TtPaaI can serve as a framework for functional multivalent display of ligands.

自然界中的许多蛋白质都需要低聚才能充分发挥其活性。此外，天然和人工蛋白质的功能可以通过改变其低聚物状态进行调整，从而开发出与生物技术相关的生物大分子。从天然来源和重新设计的低聚物支架可以改变生物大分子的低聚物状态和价态。在本报告中，我们探究了嗜热菌（Thermus thermophilus）的恒温苯乙酸降解蛋白酰基-CoA（TtPaaI）的支架化潜力。我们设计并成功制备了 TtPaaI（支架）与 galectin-7（配体）的融合蛋白，galectin-7 是一种与人类疾病有关的多功能凝集素（配体）。

引用次数: 0

The heterogeneous expression, extraction, and purification of recombinant Caldanaerobacter subterraneus subsp. tengcongensis apurine/apyrimidine endonuclease in Escherichia coli 在大肠杆菌中异构表达、提取和纯化重组的钙钛矿杆菌腾冲亚种嘌呤/嘧啶内切酶。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-09 DOI: 10.1016/j.pep.2024.106621

Wanli Guo , Dajin Wang , Wei Chen , Chuyang Rao , Yunxuan Tang , Wangfeng Li

Thermostable apurinic/apyrimidinic (AP) endonuclease (TtAP), cloned from Caldanaerobacter subterraneus subsp. tengcongensis, is an exonuclease III (Exo III) family protein with high-heat resistance, has activities of AP site endonuclease, 3′–5′ exonuclease, and 3′-nuclease, and facilitates efficient amplification of lengthy DNA fragments in PCR. However, the research of the combinant TtAP in Escherichia coli with its expression, large-scale extraction and purification of its protein was limited. In this study, we optimized the codons of TtAP gene for expression in E. coli and constructed a fusion gene encoding TtAP with a 6His tag (TtAP-6His). TtAP-6His was put into vector pET-30a⁽⁺⁾ to form the expression vector pET-30a⁽⁺⁾-TtAP-6His, and was then introduced into E. coli strain Rosetta (DE3). We established a systematic process for the extraction of TtAP protein using 5 liters of bacterial suspension, including the optimization of IPTG induction time (6 h), followed by protein extraction using enzymolysis buffers, the heat treatment of temperature (70 °C) with 60 min to remove impurity, precipitation with ammonium sulfate (55 %), protein purification with Ni-affinity chromatography, and the enzyme activities finally were determined. The purification yield of TtAP-6His ranged from 73.67 to 115.25 mg/L (47 KU/mg).

热稳定嘌呤/近嘧啶（AP）内切酶（TtAP）克隆自腾冲亚种钙单胞菌（Caldanaerobacter subterraneus subsp. tengcongensis），是一种具有高耐热性的外切酶Ⅲ（ExoⅢ）家族蛋白，具有AP位点内切酶、3'-5'外切酶和3'-核酸内切酶的活性，有助于在PCR中高效扩增冗长的DNA片段。然而，在大肠杆菌中表达 TtAP 组合、大规模提取和纯化其蛋白质的研究还很有限。在本研究中，我们优化了 TtAP 基因在大肠杆菌中的表达密码子，并构建了编码 TtAP 与 6His 标记（TtAP-6His）的融合基因。将 TtAP-6His 放入载体 pET-30a(+)，形成表达载体 pET-30a(+)-TtAP-6His，然后导入大肠杆菌菌株 Rosetta (DE3)。我们建立了一套利用 5 升菌悬液提取 TtAP 蛋白的系统流程，包括优化 IPTG 诱导时间（6 小时）、使用酶解缓冲液提取蛋白、60 分钟高温（70 ℃）热处理除杂、硫酸铵沉淀（55%）、镍亲和色谱纯化蛋白，最后测定酶活性。TtAP-6His 的纯化率为 73.67 至 115.25 mg/L（47 KU/mg）。

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引用次数: 0

Nature of recombinant human serum amyloid A1 in Escherichia coli and its preferable approach for purification 重组人血清淀粉样蛋白 A1 在大肠杆菌中的性质及其最佳纯化方法。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-05 DOI: 10.1016/j.pep.2024.106620

Saira Ahmad , Qurratulann Afza Gardner , Nisar Ahmad Shakir , Sabahat Gulzar , Naseema Azim , Muhammad Akhtar

Serum amyloid A1 (SAA1) is an apolipoprotein which is involved in amyloid A amyloidosis (AA) by forming fibrils. The process of fibrillation is still being explored and holds challenges in recombinant expression and purification of SAA1. This study deals with the preferable approach for the expression and purification of SAA1 which is normally toxic and unstable to express without using any fusion-tag. Complete soluble expression of SAA1 was obtained without the use of additional tag, in terrific broth, supplemented with 3 % ethanol at 30 °C. Soluble fraction of SAA1 was initially treated with salting-out using ammonium sulphate giving 1.5 M salt concentration to avoid SAA1 protein precipitation along with unwanted proteins. The soluble fraction of SAA1 after salting-out was purified by two individual chromatographic approaches: One anion exchange and second reverse phase chromatography. The yield of purified SAA1 was 3 times greater by anion exchange than reverse phase chromatography. MALDI-TOF analysis of purified SAA1 showed 11813 Da for intact protein and proteome analysis revealed greater than 90 % sequence coverage by MASCOT. The subunit interaction showed hexamer form at basic pH which was analyzed by size exclusion chromatography. The fibrillation activity of SAA1 was found to be 10–15 times higher in basic media at 43 °C than 37 °C. Our research demonstrates successful expression and purification of wild-type human recombinant SAA1. The cost-effective radical approach employed for purification of SAA1 is crucial for thorough protein characterization particularly, mechanisms of protein aggregation involved in amyloidosis.

血清淀粉样蛋白 A1（SAA1）是一种脂蛋白，通过形成纤维参与淀粉样 A 淀粉样变性（AA）。纤化过程仍在探索之中，这给 SAA1 的重组表达和纯化带来了挑战。SAA1 通常具有毒性且不稳定，因此在不使用任何融合标记的情况下，表达和纯化 SAA1 是一种可取的方法。在添加了 3% 乙醇的特氏肉汤中，温度为 30 °C，在不使用额外标签的情况下，获得了 SAA1 的完全可溶性表达。SAA1 的可溶性部分最初用硫酸铵盐析处理，盐浓度为 1.5 M，以避免 SAA1 蛋白与不需要的蛋白质一起沉淀。盐析后的 SAA1 可溶性部分通过两种不同的色谱法进行纯化：一种是阴离子交换法，另一种是反相色谱法。阴离子交换法纯化的 SAA1 产量是反相色谱法的 3 倍。纯化的 SAA1 的 MALDI-TOF 分析显示其完整蛋白质的含量为 11813 Da，蛋白质组分析显示 MASCOT 的序列覆盖率超过 90%。亚基相互作用在碱性 pH 下呈现六聚体形式，并通过尺寸排阻色谱法进行了分析。研究发现，SAA1 在 43 °C 的碱性介质中的纤化活性是 37 °C 的 10-15 倍。我们的研究成功地表达和纯化了野生型人类重组 SAA1。纯化 SAA1 所采用的经济有效的激进方法对于彻底鉴定蛋白质，特别是淀粉样变性所涉及的蛋白质聚集机制至关重要。

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引用次数: 0

Trehalose-6-phosphate phosphatase expression and enzymatic properties of Fusarium graminearum Fusariumgraminearum 的脱卤糖-6-磷酸磷酸酶的表达和酶特性。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2024-11-05 DOI: 10.1016/j.pep.2024.106619

Xuebiao Zhang , Le Chen , Zhong Ni , Chao Xu , Qinyan Wu , Yiqing Zhuang

This study presents an exhaustive characterization of the enzymatic attributes and structural properties of trehalose-6-phosphate phosphatase (TPP) derived from Fusarium graminearum. Enzyme activity was evaluated through a meticulously designed enzymatic assay. The findings indicate that the molecular weight of the enzyme is approximately 99.8 kDa, with an optimal reaction temperature and pH of 40 °C and 6.5, respectively. Magnesium ions (Mg²⁺) markedly enhance the enzymatic activity, resulting in a specific activity of 1.795 U/μg. Kinetic analysis revealed a K_m value of 0.96 μmol/L and a V_max of 15.79 μmol/L/min. Subsequent computational analysis elucidated the three-dimensional architecture of the enzyme and identified the binding site for the substrate trehalose-6-phosphate (T6P). T6P was found to form hydrogen bonds with TPP at residues Lys754, Arg720, His665, Glu758, and Asn756. Additionally, hydrophobic interactions were observed between T6P and residues Phe802, Ile610, Asp801, Pro752, and Gly753. The binding energy calculated for the T6P-TPP complex stood at −5.7 kcal/mol.

本研究详尽描述了从禾谷镰刀菌（Fusarium graminearum）中提取的三卤糖-6-磷酸磷酸酶（TPP）的酶属性和结构特性。通过精心设计的酶测定法对酶活性进行了评估。研究结果表明，该酶的分子量约为 99.8 kDa，最佳反应温度和 pH 值分别为 40 °C 和 6.5。镁离子（Mg2+）能显著提高酶的活性，使酶的比活达到 1.795 U/μg 。动力学分析表明，Km 值为 0.96 μmol/L，Vmax 为 15.79 μmol/L/min。随后的计算分析阐明了该酶的三维结构，并确定了底物三卤糖-6-磷酸（T6P）的结合位点。研究发现，T6P 与 TPP 在 Lys754、Arg720、His665、Glu758 和 Asn756 残基上形成氢键。此外，还观察到 T6P 与残基 Phe802、Ile610、Asp801、Pro752 和 Gly753 之间的疏水相互作用。计算得出的 T6P-TPP 复合物结合能为 -5.7 kcal/mol。

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引用次数: 0