Protein expression and purification最新文献_第7页

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

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-09-04 DOI: 10.1016/j.pep.2025.106810

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

{"title":"High-level soluble expression of human aldehyde dehydrogenase 2 in Escherichia coli achieved through lactose-mediated induction optimization","authors":"Hongxia Li, Xiong Wang, Xinye Wang, Zhang Zhang, Linmin Ran","doi":"10.1016/j.pep.2025.106810","DOIUrl":"10.1016/j.pep.2025.106810","url":null,"abstract":"<div><div>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 <em>Escherichia coli</em>. 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106810"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145004120","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

Recombinant expression and purification of Plasmodium heme detoxification protein in E. coli: Challenges and discoveries 疟原虫血红素解毒蛋白在大肠杆菌中的重组表达与纯化：挑战与发现。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-05 DOI: 10.1016/j.pep.2025.106789

Rahul Singh , Ravindra D. Makde

{"title":"Recombinant expression and purification of Plasmodium heme detoxification protein in E. coli: Challenges and discoveries","authors":"Rahul Singh , Ravindra D. Makde","doi":"10.1016/j.pep.2025.106789","DOIUrl":"10.1016/j.pep.2025.106789","url":null,"abstract":"<div><div>Heme, a toxic by-product of <em>Plasmodium</em>'s proteolytic digestion of host hemoglobin, is detoxified by the malaria parasite through its conversion into hemozoin (Hz)—the malaria pigment. This detoxification pathway is a key target for many antimalarial drugs, which aim to induce heme-mediated toxicity to the parasite. The Heme Detoxification Protein (HDP) plays a central role in heme-to-Hz transformation; however, its precise mechanism remains unclear, largely due to the absence of successful recombinant expression in a native, soluble form.</div><div>In this study, we aimed to express HDP recombinantly in its native soluble state using an <em>E. coli</em>-based system. A range of strategies were employed, including expression of orthologs, consensus sequence design, fusion to solubility-enhancing partners, co-expression with molecular chaperones, and extensive construct optimization through N-terminal truncations. Despite extensive efforts, most recombinant HDP constructs were either insoluble or formed soluble aggregates. Notably, only a single construct—with a 44-residue N-terminal truncation and a C-terminal 6-His tag (<em>HDPpf-C10</em>)—was successfully expressed in a soluble form.</div><div>Surprisingly, HDPpf-C10, although retaining domains implicated in heme binding and transformation, exhibited no detectable heme-to-Hz transformation activity. This finding highlights the essential role of the flexible-unstructured N-terminal region in mediating both heme binding and its subsequent conversion to Hz, providing new insights into HDP function and guiding future structural and mechanistic studies.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106789"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144785126","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

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

Protein expression and purification Pub Date : 2025-12-01 Epub 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

{"title":"A magnetic bead-based fluorescent substrate for sensitive assay of SARS-CoV-2 3C-like protease activity","authors":"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","doi":"10.1016/j.pep.2025.106811","DOIUrl":"10.1016/j.pep.2025.106811","url":null,"abstract":"<div><div>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 (<em>y</em> = <em>725.29x</em> − <em>52.356</em>; <em>R</em><sup><em>2</em></sup> = <em>0.998</em>) was obtained, enabling accurate quantification of the cleaved product and kinetic parameters. The assay using the designed substrate revealed a K<sub>m</sub> of 22.01 ± 3.5 μM, k<sub>cat</sub> of 0.021 s<sup>-</sup><sup>1</sup>, and catalytic efficiency (k<sub>cat</sub>/K<sub>m</sub>) of 946 M<sup>-</sup><sup>1</sup><sup>.</sup>s<sup>-</sup><sup>1</sup>. The assay showed ∼50-fold greater sensitivity compared to SDS-PAGE and the inhibitory effect of GC376 for 3CLpro was also determined, with IC<sub>50</sub> 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106811"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145016039","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

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

Protein expression and purification Pub Date : 2025-12-01 Epub 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

{"title":"High-efficient refolding and purification of recombinant human interleukin-2 from inclusion bodies","authors":"Fei Wang , Yuming Fang , Jiawei Yu , Xinyi Zhao, Yuxiao Liu, Xiaoran Jing, Jiayu Wang, Shanshan Wang, Shuo Wang, Junjun Jiang, Sheng Zhang","doi":"10.1016/j.pep.2025.106806","DOIUrl":"10.1016/j.pep.2025.106806","url":null,"abstract":"<div><div>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 <em>Escherichia coli</em>. 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106806"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144918028","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

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

Protein expression and purification Pub Date : 2025-12-01 Epub 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

{"title":"Creating a triple mutant tobacco chassis with altered cfG expression for the production of humanized therapeutic protein","authors":"Muhammad Naeem , Weihua Zhao , Tengjian Wen , Rong Han , Xuemeng Shan , Anran Xu , Lingxia Zhao","doi":"10.1016/j.pep.2025.106807","DOIUrl":"10.1016/j.pep.2025.106807","url":null,"abstract":"<div><div>Plant-based expression systems offer a promising platform for producing therapeutic glycoproteins with human-compatible glycosylation patterns. This study aimed to engineer tobacco plants (<em>Nicotiana tabacum</em> 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 <em>β-1,4-galactosyltransferase</em> (<em>GALT</em>) gene was cloned into the <em>pHB</em> vector and introduced into tobacco <em>via Agrobacterium</em>-mediated transformation. Four <em>GALT</em>-OE lines (<em>13</em><sup><em>#</em></sup><em>, 18</em><sup><em>#</em></sup><em>, 22</em><sup><em>#</em></sup> and <em>30</em><sup><em>#</em></sup>) were generated which showed significantly higher <em>GALT</em> expression, especially <em>GALT-</em>OE <em>30</em><sup><em>#</em></sup> which showed a 4.5-fold increase over wild-type (WT). Moreover, Western-blot and ELISA analyses showed that protein expression in <em>galt13</em><sup><em>#</em></sup>, and <em>galt30</em><sup><em>#</em></sup> was also increased. Triple mutants were generated by crossing the <em>GALT-</em>OE 30<sup>#</sup> line with previously developed double mutants <em>β-1,2-xylosyltransferase</em> (<em>CXT1P</em>-RNAi) and <em>α-1,3-fucosyltransferase</em> (<em>FUT4</em>-RNAi), which showed a 70 % and 80 % reduction in <em>CXT1P</em> and <em>FUT4</em> expression levels, respectively. The generated triple mutants (<em>cfG028, cfG031,</em> and <em>cfG039</em>) showed a 3.8-fold increase in <em>GALT</em> 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106807"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144966316","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

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

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-23 DOI: 10.1016/j.pep.2025.106805

Subhankar Metya , Shoaib Haidar , Anurag S. Rathore

{"title":"Efficient and economical purification platform for production of therapeutic nanobodies","authors":"Subhankar Metya , Shoaib Haidar , Anurag S. Rathore","doi":"10.1016/j.pep.2025.106805","DOIUrl":"10.1016/j.pep.2025.106805","url":null,"abstract":"<div><div>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 <em>Escherichia coli</em> (<em>E. coli)</em>. 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106805"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144893767","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

Toward reproducible PETase research: A standardized workflow for reliable enzyme production and comparison 迈向可重复的PETase研究：可靠的酶生产和比较的标准化工作流程。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-15 DOI: 10.1016/j.pep.2025.106801

Katerina Jiraskova, Jakub Ptacek, Kristyna Vydra Bousova, Jiri Vondrasek

{"title":"Toward reproducible PETase research: A standardized workflow for reliable enzyme production and comparison","authors":"Katerina Jiraskova, Jakub Ptacek, Kristyna Vydra Bousova, Jiri Vondrasek","doi":"10.1016/j.pep.2025.106801","DOIUrl":"10.1016/j.pep.2025.106801","url":null,"abstract":"<div><div>The enzymatic degradation of polyethylene terephthalate (PET) by PETases has gained significant attention as a potential solution for plastic waste management. However, the absence of a standardized protocol for PETase production across studies presents a challenge for consistent enzyme characterization and activity comparison. Variations in production methods, including expression systems and purification techniques, may contribute to discrepancies in reported PETase activities. Here, we present the development of a unified protocol for the production of wild-type and engineered <em>Is</em>PETase variants. This protocol comprises standardized expression, purification, and quality control steps to ensure reproducibility and reliability. By enabling more accurate comparisons of PETase variants and addressing inconsistencies in PETase production, this approach facilitates collaborative efforts to advance plastic degradation technologies and lays the groundwork for accelerating research in enzymatic PET degradation and its applications in plastic waste management.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106801"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144862366","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

Intracytoplasmic sperm injection-mediated strategy for the production of transgenic poultry as a bioreactor 胞浆内精子注射介导的转基因家禽生物反应器生产策略

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-18 DOI: 10.1016/j.pep.2025.106802

Shusei Mizushima

引用次数: 0

Expression of a human Gb3/CD77 synthase in insect and human cells: comparison of activity and glycosylation 人Gb3/CD77合成酶在昆虫和人细胞中的表达：活性和糖基化的比较

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-18 DOI: 10.1016/j.pep.2025.106803

Krzysztof Mikolajczyk , Katarzyna Szymczak-Kulus , Anna Bereznicka , Radoslaw Kaczmarek , Lukasz Filip Sobala , Anna Jakubiak-Augustyn , Marcin Czerwinski

{"title":"Expression of a human Gb3/CD77 synthase in insect and human cells: comparison of activity and glycosylation","authors":"Krzysztof Mikolajczyk , Katarzyna Szymczak-Kulus , Anna Bereznicka , Radoslaw Kaczmarek , Lukasz Filip Sobala , Anna Jakubiak-Augustyn , Marcin Czerwinski","doi":"10.1016/j.pep.2025.106803","DOIUrl":"10.1016/j.pep.2025.106803","url":null,"abstract":"<div><div>Glycosylation of proteins can impact their folding, stability, trafficking and enzymatic activity. Human Gb3/CD77 synthase (α1,4-galactosyltransferase, A4galt) has two occupied N-glycosylation sites. Previously, we demonstrated that the activity of recombinant enzyme relies on its N-glycosylation. In this study, we produced soluble recombinant catalytic domain of human Gb3/CD77 synthase in two expression hosts known for different glycosylation patterns: <em>Trichoplusia ni</em> insect cells (High Five) and human embryonic kidney cells (Expi293F™). The High Five cells generate short oligomannose structures, while the Expi293F™ cells synthesize complex type glycans. We evaluated the activity of High Five-derived and Expi293F™-derived enzymes, characterized the structures of their N-glycans and showed that High Five cells provide a higher amount and activity of the enzyme. Moreover, we used the Expi293F™ cells to evaluate the N- and C-terminal location of the 6xHis-tag and found that only the N-terminally tagged Expi293F™-derived enzyme demonstrated activity. In contrast, the enzyme produced in High Five cells was active despite carrying a C-terminal tag. These findings highlight the role of glycosylation pattern and tag position in the activity of human recombinant glycosyltransferase produced in different hosts.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106803"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144912957","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

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

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-20 DOI: 10.1016/j.pep.2025.106804

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

{"title":"Enhanced production of stem bromelain in Pichia pastoris by coexpression of unfolded protein response activator gene HAC1P","authors":"Sindhu Varadharaj , Jayachandran Krishnan , Mohd Imran Shah , Meenakshisundaram Sankaranarayanan","doi":"10.1016/j.pep.2025.106804","DOIUrl":"10.1016/j.pep.2025.106804","url":null,"abstract":"<div><div>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 <em>Ananas comosus</em>. 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) <em>Hac1</em> on folding and secretion of recombinant stem bromelain in <em>Pichia pastoris.</em> Stem bromelain gene (BL) from <em>Ananas comosus</em> was codon optimized and expressed in the <em>Pichia pastoris</em> X-33 host using constitutive and inducible promoters. To fold the misfolded protein aggregates in Endoplasmic Reticulum (ER), UPR regulator, <em>Hac1p</em> 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 <em>HAC1</em> was co-expressed with stem bromelain (BL). Fed batch studies were performed for both <em>pGAPαBL1</em> and <em>pGAPBLHAC1</em> 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 <em>like Kar2p, Sec 63, Pdi, Cne1</em>. The stem bromelain activity of 54 U/mL is the highest activity reported so far in the literature. The current work signifies <em>Pichia pastoris</em> as a robust platform to produce stem bromelain for various industrial applications.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106804"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144918027","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