Protein expression and purification最新文献_第7页

Adaptation of induced pluripotent stem cell technology for avian species 诱导多能干细胞技术在鸟类中的适应性研究。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-09-11 DOI: 10.1016/j.pep.2025.106814

Masafumi Katayama , Tomokazu Fukuda

Following the first report of induced pluripotent stem (iPS) cells from mouse, various mammalian-derived iPS cells have been established. In contrast, avian-derived iPS cells or iPS-like cells have been rarely reported. iPS cells can differentiate into various cell types (e.g., neural cells and hepatocytes) and proliferate indefinitely in culture. Unlike embryonic stem cells, iPS cells are generated from somatic cells, eliminating the need for embryos in their generation. Because somatic cells can be obtained from deceased individuals, iPS cell technology can be adapted for use in wild avian species, beyond its application in chickens. Our group previously reported the generation of chicken iPS cells from somatic cells using a modified-octamer-binding transcription factor 3/4 (Oct3/4), SRY-box transcription factor 2 (Sox2), Krüppel-like factor 4 (Klf4), MYC proto-oncogene (c-Myc), Nanog, and lin-28 homolog A (Lin28A). Developmental chicken eggs are a valuable resource for protein production. We may obtain valuable proteins from the chimeric developmental eggs of chickens using genome-edited or transgenic chicken iPS cells. Furthermore, our group established iPS cells derived from endangered avian species (Okinawa rail, Japanese ptarmigan, and Blakiston's fish owl) using modified-Oct3/4, Sox2, Klf4, c-Myc, Nanog, Lin28, and Klf2. Cells differentiated from iPS cells (e.g., neural cells and hepatocytes) can be used for drug testing in veterinary medicine and for evaluating sensitivity to infectious diseases and pollutants. We believe that iPS cell technology can be developed as a powerful tool for the conservation of endangered avian species.

继首次报道小鼠诱导多能干细胞（iPS）后，已经建立了各种哺乳动物来源的iPS细胞。相比之下，来自禽类的iPS细胞或iPS样细胞鲜有报道。iPS细胞可以分化成各种细胞类型（如神经细胞和肝细胞），并在培养中无限增殖。与胚胎干细胞不同，诱导多能干细胞是由体细胞产生的，因此不需要胚胎。由于体细胞可以从死亡个体中获得，iPS细胞技术可以适用于野生鸟类物种，而不仅仅是在鸡身上的应用。本小组之前报道了利用修饰的八聚体结合转录因子3/4 （Oct3/4）、sly -box转录因子2 （Sox2）、kr ppel样因子4 （Klf4）、MYC原癌基因（c-Myc）、Nanog和lin-28同源物a （Lin28A）从体细胞中生成鸡iPS细胞。发育中的鸡蛋是生产蛋白质的宝贵资源。我们可以使用基因组编辑或转基因鸡iPS细胞从鸡的嵌合发育蛋中获得有价值的蛋白质。此外，我们的团队利用修饰过的- oct3 /4、Sox2、Klf4、c-Myc、Nanog、Lin28和Klf2建立了从濒危鸟类（冲绳秧鸡、日本石鸡和Blakiston鱼鸮）中提取的iPS细胞。从诱导多能干细胞分化出来的细胞（如神经细胞和肝细胞）可用于兽医学的药物测试和评估对传染病和污染物的敏感性。我们相信iPS细胞技术可以发展成为保护濒危鸟类物种的有力工具。

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引用次数: 0

Transcriptome profiles for defining avian primordial germ cell development 定义禽原始生殖细胞发育的转录组谱。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-09-11 DOI: 10.1016/j.pep.2025.106812

Kennosuke Ichikawa, Mike J. McGrew

Avian biology has contributed to many research areas, such as sustainable protein production, endocrinology, developmental biology, neurosciences, and immunology. Primordial germ cells, lineage-restricted stem cells, are key for the conservation of genetic diversity of bird species, as well as for studying germ cell development and producing genetic models to study avian biology. Here, we review the current knowledge of developmental and fate decision processes in avian primordial germ cells focusing on insights revealed by gene expression profiling. We summarized the characteristics and fundamental pathways required for chicken primordial cell growth. In addition, we discuss the common and disparate features of PGCs from chicken compared to other avian species. These insights are valuable for researchers in germ cell biology, reproductive biotechnology, and avian genetic conservation and indicate a need for the analysis of further bird species.

鸟类生物学对可持续蛋白质生产、内分泌学、发育生物学、神经科学和免疫学等许多研究领域做出了贡献。原始生殖细胞，即谱系限制型干细胞，是保护鸟类遗传多样性、研究生殖细胞发育和建立鸟类生物学遗传模型的关键。在这里，我们回顾了目前对鸟类原始生殖细胞发育和命运决定过程的了解，重点介绍了基因表达谱揭示的见解。我们总结了鸡原始细胞生长的特点和基本途径。此外，我们还讨论了与其他鸟类相比，鸡PGCs的共同和不同特征。这些见解对生殖细胞生物学、生殖生物技术和鸟类遗传保护的研究人员有价值，并表明需要进一步分析鸟类物种。

引用次数: 0

A magnetic bead-based fluorescent substrate for sensitive assay of SARS-CoV-2 3C-like protease activity 基于磁珠的荧光底物用于sars - cov - 23c样蛋白酶活性的灵敏测定。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-09-05 DOI: 10.1016/j.pep.2025.106811

Thanh-Hoa T. Tran , Trung-Duc Nguyen , Ngoc-Nam Phan , Hang T. Ngo , Phuc-Loc Nguyen Do , Phan-Anh Le , Nho-Thai Dinh , Tuan-Nghia Phan , Hong-Loan T. Nguyen

The 3C-like protease (3CLpro) of SARS-CoV-2 is a crucial target for antiviral drugs due to its essential role in viral polyprotein processing. In this study, we designed and produced a modular fluorescent recombinant substrate (6×His-ECFP-AVLQSGFRK-EYFP), which was then immobilized on Ni-NTA magnetic beads (Ni-NTA-6×His-ECFP-AVLQSGFRK-EYFP) for the assay of 3CLpro activity. Upon cleavage at the specific AVLQ↓SG motif, the EYFP fragment was released into the supernatant and quantified via fluorescence measurement (Ex/Em = 480/528 nm). A standard curve (y = 725.29x − 52.356; R² = 0.998) was obtained, enabling accurate quantification of the cleaved product and kinetic parameters. The assay using the designed substrate revealed a K_m of 22.01 ± 3.5 μM, k_cat of 0.021 s^-¹, and catalytic efficiency (k_cat/K_m) of 946 M^-¹^.s^-¹. The assay showed ∼50-fold greater sensitivity compared to SDS-PAGE and the inhibitory effect of GC376 for 3CLpro was also determined, with IC₅₀ of 0.88 μM. Since the modular substrate design allows for substitution of the N-terminal domain and cleavage motif, our development of the substrate and assay could be expanded to other high-specificity proteases.

SARS-CoV-2的3c样蛋白酶（3CLpro）在病毒多蛋白加工过程中发挥重要作用，是抗病毒药物的重要靶点。在本研究中，我们设计并制作了模块化荧光重组底物（6×His-ECFP-AVLQSGFRK-EYFP），然后将其固定在Ni-NTA磁珠（Ni-NTA-6×His-ECFP-AVLQSGFRK-EYFP）上，用于测定3CLpro的活性。在特定的AVLQ↓SG基序上切割后，EYFP片段被释放到上清中，并通过荧光测量（Ex/Em = 480/528 nm）进行定量。得到标准曲线（y = 725.29x - 52.356; R2 = 0.998），可准确定量裂解产物及动力学参数。实验结果表明，该底物的Km为22.01±3.5 μM， kcat为0.021 s-1，催化效率（kcat/Km）为946 m -1 s-1。与SDS-PAGE相比，该方法的灵敏度提高了约50倍，并且还确定了GC376对3CLpro的抑制作用，IC50为0.88 μM。由于模块化底物设计允许替换n端结构域和切割基序，因此我们的底物开发和检测可以扩展到其他高特异性蛋白酶。

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引用次数: 0

High-level soluble expression of human aldehyde dehydrogenase 2 in Escherichia coli achieved through lactose-mediated induction optimization 通过乳糖介导的诱导优化，实现了人醛脱氢酶2在大肠杆菌中的高可溶性表达

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-09-04 DOI: 10.1016/j.pep.2025.106810

Hongxia Li, Xiong Wang, Xinye Wang, Zhang Zhang, Linmin Ran

Aldehyde dehydrogenase 2 (ALDH2) plays a critical role in ethanol metabolism by converting toxic acetaldehyde to acetate. To investigate its functional mechanisms and potential therapeutic applications for alcohol-related diseases, heterologous expression of ALDH2 is essential. However, ALDH2 often forms inclusion bodies when expressed in Escherichia coli. In this work, the solubility of ALDH2 was enhanced by systematic optimization of expression conditions using IPTG and lactose as respective inducers. Under optimized conditions, the media yield of ALDH2 induced by IPTG and lactose reached 44.5 ± 1.3 and 48.7 ± 1.2 μg/mL respectively, representing 7.1- and 7.8-fold improvements over unoptimized conditions. Enzymatic characterization revealed that purified ALDH2 exhibited optimal activity of 9.7 U/mL at 37 °C and pH 8.0. This research demonstrates that optimizing expression conditions is an effective strategy to enhance the solubility of recombinant enzymes, while providing a practical solution for other enzymes prone to inclusion body formation.

醛脱氢酶2 （ALDH2）通过将有毒的乙醛转化为乙酸，在乙醇代谢中起关键作用。为了研究其功能机制及其在酒精相关疾病中的潜在治疗应用，异源表达ALDH2是必不可少的。然而，ALDH2在大肠杆菌中表达时往往形成包涵体。本研究以IPTG和乳糖为诱导剂，通过系统优化表达条件，提高了ALDH2的溶解度。优化条件下，IPTG和乳糖诱导ALDH2的培养基产率分别达到44.5±1.3和48.7±1.2 μg/mL，分别比未优化条件提高7.1和7.8倍。酶学鉴定表明，纯化后的ALDH2在37℃、pH 8.0条件下的酶活性为9.7 U/mL。本研究表明，优化表达条件是提高重组酶溶解度的有效策略，同时也为其他易形成包涵体的酶提供了切实可行的解决方案。

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引用次数: 0

Enhanced performance of Thermococcus kodakarensis KOD1 polymerase in PCR via fusion to Sulfolobus tokodaii Sto7d 通过融合tokodaisulfolobus Sto7d提高柯达热球菌KOD1聚合酶的PCR性能

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-31 DOI: 10.1016/j.pep.2025.106809

Leheng Chen , Dawei Fu

The DNA polymerase from Thermococcus kodakarensis KOD1 (KOD) is widely utilized in polymerase chain reaction (PCR) due to its high processivity and fidelity. However, like many other B-family DNA polymerases, it faces limitations in extension efficiency, amplicon length, and resistance to PCR inhibitors. In order to further enhance its capability, novel mutants were engineered by fusing a 7 kDa nonspecific double-stranded DNA (dsDNA)-binding protein from Sulfolobus tokodaii (Sto7d) to the C-terminus of KOD via distinct peptide linkers, resulting in a set of KOD-Sto7d polymerase variants. These constructs were expressed, purified, and characterized. Among the variants, KOD-GT4G-Sto7d exhibited the best PCR performance and was selected as the representative variant for subsequent assays. Compared with wild-type KOD (KOD-WT), KOD-Sto7d demonstrated significantly improved extension efficiency that successfully amplified 7 kb targets with only 10 s elongation time, increased salt tolerance up to 120 mM NaCl for 2 kb targets, and an improved capacity to amplify long DNA fragments up to 10 kb within 4 min. In comparison with a commercially available KOD mutant fused to a dsDNA-binding protein (Sso7d from Saccharolobus solfataricus) at its C-terminus (KOD-Sso7d), KOD-Sto7d demonstrated greater salt tolerance and sensitivity. These results suggest that KOD-Sto7d is a robust polymerase suitable for time-saving and high-demanding PCR.

kodakarensis热球菌（Thermococcus KOD1， KOD）的DNA聚合酶因其高精密度和保真度被广泛应用于聚合酶链反应（polymerase chain reaction， PCR）中。然而，像许多其他b家族DNA聚合酶一样，它在延伸效率、扩增子长度和对PCR抑制剂的抗性方面存在局限性。为了进一步增强其能力，我们将tokodaii Sulfolobus （Sto7d）中一个7 kDa的非特异性双链DNA （dsDNA）结合蛋白通过不同的肽连接体融合到KOD的c端，从而构建了一组KOD-Sto7d聚合酶变体。这些结构被表达、纯化和表征。其中，KOD-GT4G-Sto7d表现出最好的PCR性能，并被选为后续检测的代表性变异。与野生型KOD （KOD- wt）相比，KOD- sto7d扩增效率显著提高，在10 s的延伸时间内扩增出7 kb的目标，对2 kb目标的耐盐性提高到120 mM NaCl，在4 min内扩增10 kb长的DNA片段的能力也有所提高。与市售的在其c端融合dsdna结合蛋白（来自Saccharolobus solfataricus的Sso7d）的KOD突变体（KOD-Sso7d）相比，KOD- sto7d表现出更强的耐盐性和敏感性。这些结果表明KOD-Sto7d是一种强大的聚合酶，适合于节省时间和高要求的PCR。

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引用次数: 0

Advancing recombinant antibody production in E. coli: Optimization of expression and purification via dual GFP promoter and imaging technology 推进重组抗体在大肠杆菌中的生产：利用双GFP启动子和成像技术优化表达和纯化。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-28 DOI: 10.1016/j.pep.2025.106808

Anttoni Korkiakoski, Sami Oksanen, Tuomas Huovinen

Fed-batch fermentation results in high recombinant protein titers in limited culture volumes. Therefore, it is the preferred operation mode in the bioprocess industry. Optimizing feeding, induction, and harvest timing is a significant time-consuming challenge in bioprocessing complicated by the fact that expressed target protein is rarely detectable in real-time. In this study, the construction of an online sensor is described integrating a dual GFP promoter construct, a blue LED and a Raspberry Pi camera for real-time monitoring of recombinant antibody expression in Escherichia coli. The dual promoter construct allows simultaneous expression of GFP in the cytoplasm and the recombinant antibody in the periplasm, enabling the use of GFP fluorescence as a proxy for protein yield. GFP fluorescence correlated with Fab and nanobody expression over time and the relative quantity of fluorescence predicted the extent of induction. In nanobody fed-batch fermentations, the decreasing rate of dGFP/dt was a valuable parameter for identifying the optimal harvest point, minimizing excessive incubation time and reducing nanobody leakage into the medium. It was further demonstrated that quantitation of pixel values from RGB images captured with a Raspberry Pi 8 MP camera in the flow cell resulted in equal sensitivity for GFP detection as that achieved with a μPMT and photodiode sensors. The 3D-printable GFP sensor station is a valuable tool for process optimization and for educating bioprocess engineering students through real-time visualization of promoter activation.

补料分批发酵结果高重组蛋白滴度在有限的培养体积。因此，它是生物加工行业的首选操作模式。在生物加工中，优化饲养、诱导和收获时间是一个非常耗时的挑战，因为表达的目标蛋白很少能实时检测到。在这项研究中，描述了一个在线传感器的构建，集成了双GFP启动子结构，一个蓝色LED和一个树莓派相机，用于实时监测重组抗体在大肠杆菌中的表达。双启动子结构允许在细胞质中同时表达GFP和在细胞质中同时表达重组抗体，从而可以使用GFP荧光作为蛋白质产量的代理。随着时间的推移，GFP荧光与Fab和纳米体的表达相关，荧光的相对数量预测了诱导程度。在纳米体分批补料发酵中，dGFP/dt的下降速率是确定最佳收获点、减少过多孵育时间和减少纳米体泄漏到培养基中的一个有价值的参数。进一步证明，用树莓派8 MP相机在流池中捕获的RGB图像的像素值的定量导致GFP检测的灵敏度与μPMT和光电二极管传感器相同。3d打印GFP传感器站是过程优化和通过实时可视化启动子激活教育生物过程工程专业学生的宝贵工具。

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引用次数: 0

Creating a triple mutant tobacco chassis with altered cfG expression for the production of humanized therapeutic protein 创建一个改变cfG表达的三突变烟草底盘，用于生产人源化治疗蛋白。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-27 DOI: 10.1016/j.pep.2025.106807

Muhammad Naeem , Weihua Zhao , Tengjian Wen , Rong Han , Xuemeng Shan , Anran Xu , Lingxia Zhao

Plant-based expression systems offer a promising platform for producing therapeutic glycoproteins with human-compatible glycosylation patterns. This study aimed to engineer tobacco plants (Nicotiana tabacum cv. Yunyan 87) to modify glycosylation pathways for the production of glycoproteins with reduced immunogenicity, enhancing their potential for therapeutic applications. To achieve this, a 1257 bp fragment of the human β-1,4-galactosyltransferase (GALT) gene was cloned into the pHB vector and introduced into tobacco via Agrobacterium-mediated transformation. Four GALT-OE lines (13^#, 18^#, 22^# and 30^#) were generated which showed significantly higher GALT expression, especially GALT-OE 30^# which showed a 4.5-fold increase over wild-type (WT). Moreover, Western-blot and ELISA analyses showed that protein expression in galt13^#, and galt30^# was also increased. Triple mutants were generated by crossing the GALT-OE 30^# line with previously developed double mutants β-1,2-xylosyltransferase (CXT1P-RNAi) and α-1,3-fucosyltransferase (FUT4-RNAi), which showed a 70 % and 80 % reduction in CXT1P and FUT4 expression levels, respectively. The generated triple mutants (cfG028, cfG031, and cfG039) showed a 3.8-fold increase in GALT expression, and corresponding glycoprotein modifications at the protein level. This study establishes a foundation for the large-scale production of low-immunogenic recombinant glycoproteins with enhanced therapeutic efficacy using a tobacco-based system.

基于植物的表达系统为生产具有人类相容糖基化模式的治疗性糖蛋白提供了一个有前途的平台。本研究旨在改造烟草植物（Nicotiana tabacum cv.）。Yunyan 87)修饰糖基化途径以产生免疫原性降低的糖蛋白，增强其治疗应用的潜力。为此，将人β-1,4-半乳糖转移酶（GALT）基因的1257 bp片段克隆到pHB载体中，并通过农杆菌介导的转化将其导入烟草。得到的4个GALT- oe株系（13#、18#、22#和30#）的GALT表达量显著高于野生型（WT），其中GALT- oe 30#的表达量比野生型（WT）高出4.5倍。此外，western-blot和ELISA分析显示，galt13#和galt30#蛋白表达也升高。将GALT-OE 30#系与先前培养的双突变体β-1,2-木糖基转移酶（CXT1P- rnai）和α-1,3- focusyltransferase （FUT4- rnai）杂交产生三突变体，结果显示CXT1P和FUT4的表达水平分别降低了70%和80%。生成的三突变体（cfG028、cfG031和cfG039） GALT表达增加3.8倍，蛋白水平上出现相应的糖蛋白修饰。本研究为大规模生产低免疫原性重组糖蛋白奠定了基础，并利用烟草为基础的系统提高了治疗效果。

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引用次数: 0

High-efficient refolding and purification of recombinant human interleukin-2 from inclusion bodies 包涵体中重组人白细胞介素-2的高效重折叠和纯化

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-23 DOI: 10.1016/j.pep.2025.106806

Fei Wang , Yuming Fang , Jiawei Yu , Xinyi Zhao, Yuxiao Liu, Xiaoran Jing, Jiayu Wang, Shanshan Wang, Shuo Wang, Junjun Jiang, Sheng Zhang

Human interleukin-2 (hIL-2) serves as a crucial cytokine in the treatment of cancer and autoimmune disorders. Nevertheless, the advancement of research and clinical applications involving this cytokine has been hindered by the constraints associated with the production of recombinant human interleukin-2 (rhIL-2). This study presents a scalable and robust purification protocol for rhIL-2 derived from inclusion bodies (IBs) in Escherichia coli. Our results indicate that microfiltration-based method could improve the purity of the denatured IBs effectively, and various refolding conditions were assessed to improve the recovery of refolded rhIL-2, resulting in an increase in the refolding yield from 15 % to 45 %. Subsequently, purification through three-column chromatography could refine the refolded rhIL-2 efficiently. Ultimately, the robustness of the purification process is substantiated by three consecutive scale-up experiments, achieving a productivity of 4 mg rhIL-2/g cell pellets, alongside high product purity and significant product activity.

人类白细胞介素-2 （il -2）在癌症和自身免疫性疾病的治疗中起着至关重要的细胞因子作用。然而，涉及该细胞因子的研究和临床应用的进展一直受到重组人白细胞介素-2 （rhIL-2）产生相关限制的阻碍。本研究提出了一种可扩展且可靠的纯化方案，用于纯化大肠杆菌包涵体（IBs）中衍生的rhIL-2。我们的研究结果表明，基于微滤的方法可以有效地提高变性IBs的纯度，并评估了不同的重折叠条件，以提高重折叠的rhIL-2的回收率，使重折叠收率从15%提高到45%。随后，通过三柱层析纯化，可以有效地提纯重组的rhIL-2。最终，纯化过程的稳健性通过三个连续的放大实验得到证实，实现了4 mg rhIL-2/g细胞颗粒的生产率，同时产品纯度高，产品活性显著。

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引用次数: 0

Efficient and economical purification platform for production of therapeutic nanobodies 高效、经济的治疗性纳米体生产纯化平台

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-23 DOI: 10.1016/j.pep.2025.106805

Subhankar Metya , Shoaib Haidar , Anurag S. Rathore

Single-domain antibodies, known as nanobodies, have arisen as an exciting class of biomolecules with applications ranging from diagnostics to numerous therapies. Due to their small size, enhanced stability, and reduced production complexity compared to traditional monoclonal antibodies, nanobodies present an economically attractive option with considerable commercial promise. In this paper, we describe a robust, efficient, and cost-effective downstream purification platform for production of nanobody-based biotherapeutics produced in Escherichia coli (E. coli). A peptide fused with the nanobody has been taken as a model. Our process utilizes periplasmic extraction to obtain the soluble product, followed by a mild acid precipitation step to remove impurities, and a single multimodal chromatography step, with an overall yield of 82.6 % and a purity exceeding 95 %. An extensive cost analysis indicated a production cost of about $17.7 per gram, far cheaper than the conventional mAb-based biopharmaceuticals. The proposed platform can facilitate commercialization of therapeutic nanobodies.

单域抗体，被称为纳米体，已经成为一类令人兴奋的生物分子，其应用范围从诊断到多种治疗。与传统的单克隆抗体相比，纳米抗体体积小，稳定性强，生产复杂性低，具有经济上的吸引力，具有相当大的商业前景。在本文中，我们描述了一个强大的、高效的、具有成本效益的下游纯化平台，用于生产由大肠杆菌（E. coli）生产的基于纳米体的生物疗法。以与纳米体融合的肽为模型。我们的工艺采用周质萃取获得可溶性产物，然后是温和的酸沉淀步骤以去除杂质，然后是单一的多模态色谱步骤，总收率为82.6%，纯度超过95%。一项广泛的成本分析表明，每克生产成本约为17.7美元，远低于传统的单克隆抗体生物制药。该平台可以促进治疗性纳米体的商业化。

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引用次数: 0

Enhanced production of stem bromelain in Pichia pastoris by coexpression of unfolded protein response activator gene HAC1P 未折叠蛋白反应激活因子基因HAC1P的共表达促进毕赤酵母茎菠萝蛋白酶的产生

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-20 DOI: 10.1016/j.pep.2025.106804

Sindhu Varadharaj , Jayachandran Krishnan , Mohd Imran Shah , Meenakshisundaram Sankaranarayanan

Expression of heterologous proteins and metabolites at high titers mounts several stress responses on the recombinant host. Stem Bromelain is a cysteine protease enzyme present in the stem and fruit of the pineapple plant Ananas comosus. The enzyme has a broad range of industrial application ranging from food, nutraceutical, cosmetic and pharmaceutical. The current work aims to study the effect of unfolded protein response regulator (UPR) Hac1 on folding and secretion of recombinant stem bromelain in Pichia pastoris. Stem bromelain gene (BL) from Ananas comosus was codon optimized and expressed in the Pichia pastoris X-33 host using constitutive and inducible promoters. To fold the misfolded protein aggregates in Endoplasmic Reticulum (ER), UPR regulator, Hac1p was co-expressed with bromelain under constitutive expression by GAP promoter. Shake flask studies resulted in a 2-fold increase in the protease activity of 4 U/mL when HAC1 was co-expressed with stem bromelain (BL). Fed batch studies were performed for both pGAPαBL1 and pGAPBLHAC1 clones in 3.7 L KLF bioreactor under glycerol limited condition and highest activity of 8 U/mL and 54 U/mL were obtained respectively. Gene expression studies of the major genes in folding and secretion pathway has shown that the activation of UPR has resulted in upregulation of major chaperones like Kar2p, Sec 63, Pdi, Cne1. The stem bromelain activity of 54 U/mL is the highest activity reported so far in the literature. The current work signifies Pichia pastoris as a robust platform to produce stem bromelain for various industrial applications.

高滴度表达外源蛋白和代谢物会对重组宿主产生多种应激反应。菠萝蛋白酶是一种半胱氨酸蛋白酶，存在于菠萝植物Ananas comosus的茎和果实中。该酶具有广泛的工业应用范围，从食品，保健品，化妆品和制药。本研究旨在研究未折叠蛋白反应调节因子（UPR） Hac1对毕氏酵母中重组茎菠萝蛋白酶折叠和分泌的影响。利用组成型启动子和诱导型启动子在毕赤酵母X-33宿主中进行了菠萝蛋白酶基因（BL）的密码子优化和表达。为了折叠UPR调控因子内质网（ER）中错误折叠的蛋白聚集体，Hac1p在GAP启动子的组成表达下与菠萝蛋白酶共表达。摇瓶研究结果表明，当HAC1与茎菠萝蛋白酶（BL）共表达时，蛋白酶活性增加了2倍，为4 U/mL。在3.7 L KLF生物反应器中，在甘油限制条件下，对pgapap α bl1和pGAPBLHAC1克隆进行了批量投料研究，最高活性分别为8 U/mL和54 U/mL。折叠和分泌通路主要基因的基因表达研究表明，UPR的激活导致Kar2p、Sec 63、Pdi、Cne1等主要伴侣蛋白上调。茎菠萝蛋白酶活性为54 U/mL，是迄今为止文献报道的最高活性。目前的工作表明毕赤酵母作为一个强大的平台生产茎菠萝蛋白酶用于各种工业应用。

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引用次数: 0