Protein expression and purification最新文献_第2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

Protein expression and purification Pub Date : 2025-09-11 DOI: 10.1016/j.pep.2025.106814

Masafumi Katayama , Tomokazu Fukuda

{"title":"Adaptation of induced pluripotent stem cell technology for avian species","authors":"Masafumi Katayama , Tomokazu Fukuda","doi":"10.1016/j.pep.2025.106814","DOIUrl":"10.1016/j.pep.2025.106814","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106814"},"PeriodicalIF":1.2,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145058524","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

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

Protein expression and purification Pub Date : 2025-09-11 DOI: 10.1016/j.pep.2025.106812

Kennosuke Ichikawa, Mike J. McGrew

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 : 2025-09-05 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":"2025-09-05","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-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 : 2025-09-04 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":"2025-09-04","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

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

Protein expression and purification Pub Date : 2025-08-31 DOI: 10.1016/j.pep.2025.106809

Leheng Chen , Dawei Fu

{"title":"Enhanced performance of Thermococcus kodakarensis KOD1 polymerase in PCR via fusion to Sulfolobus tokodaii Sto7d","authors":"Leheng Chen , Dawei Fu","doi":"10.1016/j.pep.2025.106809","DOIUrl":"10.1016/j.pep.2025.106809","url":null,"abstract":"<div><div>The DNA polymerase from <em>Thermococcus kodakarensis</em> 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 <em>Sulfolobus tokodaii</em> (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 <em>Saccharolobus solfataricus</em>) 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106809"},"PeriodicalIF":1.2,"publicationDate":"2025-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144932935","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

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

Protein expression and purification Pub Date : 2025-08-28 DOI: 10.1016/j.pep.2025.106808

Anttoni Korkiakoski, Sami Oksanen, Tuomas Huovinen

{"title":"Advancing recombinant antibody production in E. coli: Optimization of expression and purification via dual GFP promoter and imaging technology","authors":"Anttoni Korkiakoski, Sami Oksanen, Tuomas Huovinen","doi":"10.1016/j.pep.2025.106808","DOIUrl":"10.1016/j.pep.2025.106808","url":null,"abstract":"<div><div>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 <em>Escherichia coli</em>. 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106808"},"PeriodicalIF":1.2,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144966270","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 : 2025-08-27 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":"2025-08-27","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

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 : 2025-08-23 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":"2025-08-23","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

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 : 2025-08-23 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":"2025-08-23","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

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 : 2025-08-20 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":"2025-08-20","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