Protein expression and purification最新文献

Discovery and development of bispecific antibodies. 双特异性抗体的发现和发展。

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.2025.106787

Tetsuya Wakabayashi, Taichi Kuramochi

{"title":"Discovery and development of bispecific antibodies.","authors":"Tetsuya Wakabayashi, Taichi Kuramochi","doi":"10.1016/j.pep.2025.106787","DOIUrl":"10.1016/j.pep.2025.106787","url":null,"abstract":"<p><p>Bispecific antibodies (BsAbs) represent a significant breakthrough in antibody-based therapeutics, offering the unique capability to engage two distinct targets simultaneously. BsAbs are expected to exert therapeutic effects that are unattainable with conventional antibody drugs. Specifically, they are being developed for use in intercellular bridging, proximity effects, dual target inhibition, and cell targeting dependent on two antigen types. In recent years, antibody drug discovery has made progress by taking advantage of this dual-targeting ability, and bispecific antibodies have been launched across multiple therapeutic areas. These include antitumor drugs intended to enhance T-cell killing activity and inhibit growth factors, drugs that mimic blood coagulation factor functions, and angiogenesis inhibitors. This review highlights the pivotal technological advancements that have overcome the manufacturing challenges associated with BsAbs, enabling the development of pharmaceutical-grade products. We use emicizumab as a case study to illustrate these developments. Particular emphasis is placed on the critical synergy between antibody engineering technology and protein purification technologies, which has played a crucial role in the successful production of BsAbs. Furthermore, we discuss recent innovations in affinity chromatography, specifically the development of alkaline-resistant Protein L resins that have significantly improved commercial production processes. We examine the unique affinity behaviors of these resins and their impact on BsAb purification. This comprehensive review aims to provide researchers and industry professionals with a thorough understanding of the current landscape and future potential of bispecific antibodies in therapeutic applications, highlighting both technical challenges and innovative solutions in this rapidly evolving field.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106787"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144776016","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

Solubilization and refolding of inclusion bodies by detergents. 洗涤剂对包裹体的增溶和再折叠作用。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-07 DOI: 10.1016/j.pep.2025.106791

Tsutomu Arakawa, Teruo Akuta, Daisuke Ejima, Kouhei Tsumoto

{"title":"Solubilization and refolding of inclusion bodies by detergents.","authors":"Tsutomu Arakawa, Teruo Akuta, Daisuke Ejima, Kouhei Tsumoto","doi":"10.1016/j.pep.2025.106791","DOIUrl":"10.1016/j.pep.2025.106791","url":null,"abstract":"<p><p>Recombinant proteins play many important roles in development of biological reagents and biopharmaceuticals. Here, we will mainly review refolding of recombinant proteins when expressed in inclusion bodies, although strategies to enhance soluble expression are described as an alternative to refolding inclusion bodies. These strategies include, but not limited to, adding chemical chaperones in cell culture media, modifying cell lysis buffer and using solubility-enhacing fusion tags. Another solubility enhancement was to generate lipid complex for membrane proteins that form insoluble proteins without lipid. Among various solubilization and refolding technologies, those using denaturant, alkaline pH and pressure are also desribed, while we focus on solubilization and refolding using detergents, which are effective and cost-friendly. Sodium dodecylsulfate, lauroyl-glutamate, sarkosyl and cetyltrimethylammonium have been extensively used, as summarized in this review. Slow or step-wise removal of denaturants or ionic detergents used to solubilize appears to play a critical role in successful refolding by maintaining the solubility of proteins during refolding. In alkaline refolding, slow pH adjustment also helps maintain protein solubility. In pressure refolding, small amount of guanidine hydrochloride assisted refolding.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106791"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144812236","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

Secretory expression and fermentation optimization of recombinant porcine epidermal growth factor in Saccharomyces cerevisiae 重组猪表皮生长因子在酿酒酵母中的分泌表达及发酵优化。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-10-08 DOI: 10.1016/j.pep.2025.106827

Jie Liu , Wenjun Xu , Bingbing Xia , Yanfei He , Shiping Huang , Jun Zhao

{"title":"Secretory expression and fermentation optimization of recombinant porcine epidermal growth factor in Saccharomyces cerevisiae","authors":"Jie Liu , Wenjun Xu , Bingbing Xia , Yanfei He , Shiping Huang , Jun Zhao","doi":"10.1016/j.pep.2025.106827","DOIUrl":"10.1016/j.pep.2025.106827","url":null,"abstract":"<div><div>Porcine epidermal growth factor (PoEGF) promotes intestinal epithelial development and enhances immune function in weaned piglets, offering potential as an alternative to in-feed antibiotics. However, large-scale application of recombinant PoEGF (rPoEGF) is hindered by high production costs and complex purification requirements. In this study, we constructed a recombinant <em>Saccharomyces cerevisiae</em> INVSc1 strain capable of expressing and secreting rPoEGF through the integration of an α-factor signal peptide-PoEGF-6 × His expression cassette. Expression was confirmed by SDS-PAGE and Western blot analysis, and bioactivity was validated using a BALB/c 3T3 fibroblast proliferation assay, yielding a maximum mitogenic activity of 10,963 U/mL. To improve production efficiency, a two-stage fermentation strategy was developed and optimized in a 4-L bioreactor. The process consisted of a glucose-based fed-batch growth phase followed by galactose-induced expression under optimized conditions (25 °C, pH 5.0, DO 30 %). This approach enabled high-cell-density cultivation (OD<sub>600</sub> ≈ 71.5) and achieved a final secreted protein yield of 30.2 mg/L. Notably, the culture containing active rPoEGF could be directly applied as a functional feed additive without the need for downstream purification. These results provide a practical, scalable, and cost-effective approach for producing bioactive rPoEGF and support its future application in swine production as a safe, antibiotic-free growth promoter.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106827"},"PeriodicalIF":1.2,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145259007","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

Circular dichroism spectroscopy in protein engineering and pharmaceutical development: Applications in structural characterization and quality assessment 圆二色光谱在蛋白质工程和药物开发中的应用：结构表征和质量评价。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-10-08 DOI: 10.1016/j.pep.2025.106826

Taiji Oyama, Satoko Suzuki, Ken-ichi Akao

{"title":"Circular dichroism spectroscopy in protein engineering and pharmaceutical development: Applications in structural characterization and quality assessment","authors":"Taiji Oyama, Satoko Suzuki, Ken-ichi Akao","doi":"10.1016/j.pep.2025.106826","DOIUrl":"10.1016/j.pep.2025.106826","url":null,"abstract":"<div><div>Circular dichroism (CD) spectroscopy is a rapid and versatile method for assessing the higher-order structures of proteins and peptides. Far-UV CD enables quantitative evaluation of secondary structures, while near-UV CD provides sensitive fingerprints of tertiary organization. With advances in recombinant protein expression, CD spectroscopy has become widely used for the characterization of novel generated proteins. This review focuses on the applications of CD spectroscopy in protein engineering and pharmaceutical sciences. Examples include strategies for reliable measurements with limited sample quantities and quality assessments such as lot-to-lot comparisons and biosimilar evaluations. CD spectroscopy also serves as a valuable tool for detecting conformational changes associated with protein–protein and protein–drug interactions, as well as for evaluating proteins and peptides in membrane-mimetic environments. Obtaining reliable CD spectra requires careful selection of buffers, water-miscible solvents, and excipients. Here, we summarize their properties and propose practical criteria for their selection to ensure high-quality CD measurements.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106826"},"PeriodicalIF":1.2,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145259018","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

Modulation of N-glycosylation in bispecific antibody biosimilars through combined modulators 通过联合调节剂调节双特异性抗体生物类似药的n -糖基化。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-09-26 DOI: 10.1016/j.pep.2025.106825

Xu Shengnan , Wang Pan , Wang Xiaofei, Liu Xiaojing, Li Guozhu, Qin Guohong, Xu Dan

{"title":"Modulation of N-glycosylation in bispecific antibody biosimilars through combined modulators","authors":"Xu Shengnan , Wang Pan , Wang Xiaofei, Liu Xiaojing, Li Guozhu, Qin Guohong, Xu Dan","doi":"10.1016/j.pep.2025.106825","DOIUrl":"10.1016/j.pep.2025.106825","url":null,"abstract":"<div><div>To ensure a high degree of similarity between biosimilars and reference drugs, it is crucial to perform comprehensive characterization and maintain rigorous control over glycosylation processes. Here,we aimed to optimize the glycosylation profile of a biosimilar of a bispecific antibody (BsAb) to closely resemble that of the reference drug through the synergistic use of glycosylation modulators. To identify the strongest modulators and appropriate concentration ranges, we first examined the effects of different concentrations of galactose (Gal) and manganese chloride (MnCl<sub>2</sub>) on the galactosylation rate in Shake Flask, as well as the influence of tris(hydroxymethyl)aminomethane (Tris) on the incorporation of mannose and fucose in 2 L Bioreactor. Importantly, the concurrent use of Tris and galactose did not result in any interaction effects on N-glycan modifications and had no detrimental impact on cell growth, metabolism, antibody charge variants or purity. In conclusion, The concurrent use of 0.75 mM Tris and 8 mM galactose yields a glycosylation profile of Bs-mAb1 that is highly comparable to that of the reference drug, thereby providing an effective strategy for optimizing glycosylation in biosimilars. These findings provide significant insights into the regulation of glycosylation in the production of therapeutic monoclonal antibodies and may contribute to enhancing the consistency and therapeutic performance of biosimilars.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106825"},"PeriodicalIF":1.2,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145186603","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

Biochemical characterization of histidinol dehydrogenase from the human pathogen Neisseria gonorrhoeae 淋病奈瑟菌组氨酸二醇脱氢酶的生化特性研究。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-09-26 DOI: 10.1016/j.pep.2025.106824

Shuping Fei , Jiatong Zhao , Wenwen Wu , Chaoqun Lian , Wanggang Tang

{"title":"Biochemical characterization of histidinol dehydrogenase from the human pathogen Neisseria gonorrhoeae","authors":"Shuping Fei , Jiatong Zhao , Wenwen Wu , Chaoqun Lian , Wanggang Tang","doi":"10.1016/j.pep.2025.106824","DOIUrl":"10.1016/j.pep.2025.106824","url":null,"abstract":"<div><div>Histidinol dehydrogenase (HisD, E.C. 1.1.1.23), encoded by the <em>hisD</em> gene and catalyzing the final two steps in the <span>l</span>-histidine biosynthesis, has emerged as a promising antibacterial target for several human pathogens, such as <em>Brucella suis</em> and <em>Mycobacterium tuberculosis</em>. Herein, biochemical properties on recombinant HisD from the human pathogen <em>Neisseria gonorrhoeae</em> (<em>Ng</em>HisD) were characterized in detail. SDS-PAGE and size-exclusion chromatography revealed that recombinant <em>Ng</em>HisD is a homodimer (∼105 kDa native size; ∼47 kDa subunit). Kinetic analysis identified this protein as a highly selective NAD<sup>+</sup>-dependent enzyme (specific activity: 17.3 ± 0.4 U mg<sup>−1</sup>), exhibiting minimal activity with NADP<sup>+</sup>. The <em>K</em><sub>M</sub> values for NAD<sup>+</sup> and <span>l</span>-histidinol were 0.9 ± 0.3 mM and 45 ± 6 μM, respectively. Maximal activity occurred at pH 9.5 and 45 °C in the presence of Mn<sup>2+</sup>. Heat-inactivation experiment showed that rapid inactivation began to occur following incubation at 45 °C for 30 min. The metal ions coordinated by the enzyme were not sequestered by 10 mM EDTA but were efficiently chelated by 1,10-phenanthroline. The recombinant <em>Ng</em>HisD lost 85 % activity in the presence of 1 mM 1,10-phenantroline. Our study not only establishes a foundation for investigating the physiological function of <em>Ng</em>HisD <em>in vivo</em> but also uncovers the absence of the canonical histidine biosynthesis pathway in <em>Neisseria cinerea</em>.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106824"},"PeriodicalIF":1.2,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145186531","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

Effective and robust separation of half-antibody byproduct in bispecific antibody purification by Sartobind Rapid A Protein A membrane chromatography Sartobind快速A蛋白A膜层析纯化双特异性抗体时半抗体副产物的有效和稳健分离。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-09-25 DOI: 10.1016/j.pep.2025.106823

Gaoya Yuan, Meng Qu, Yifeng Li

{"title":"Effective and robust separation of half-antibody byproduct in bispecific antibody purification by Sartobind Rapid A Protein A membrane chromatography","authors":"Gaoya Yuan, Meng Qu, Yifeng Li","doi":"10.1016/j.pep.2025.106823","DOIUrl":"10.1016/j.pep.2025.106823","url":null,"abstract":"<div><div>Half-antibody is a common byproduct associated with the production of asymmetric bispecific antibody (bsAb). We previously demonstrated that Protein A column chromatography can effectively separate this byproduct from the bsAb product, as these two species exhibit different binding valencies (half-antibody and intact bsAb contain one and two Protein A binding sites, respectively). Specifically, half-antibody, which binds weaker, can be selectively removed by an appropriate wash prior to elution of the target bsAb. However, the performance of this wash step is sensitive to the loading density, making the process unrobust to variations in harvest titer. In the current study, we demonstrate that Sartobind Rapid A Protein A membrane effectively reduces half-antibody from approximately 16.5% (in the load) to 0.1% (in the eluate). In addition, using Protein A membrane also improves process robustness by avoiding the impact of loading density variation on performance. Thus, for bsAb purification where half-antibody removal is required, Protein A membrane is a better choice than resin-based Protein A column.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106823"},"PeriodicalIF":1.2,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145182205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Expression and purification of C14orf119 and generation of its polyclonal antibody C14orf119的表达纯化及其多克隆抗体的制备。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-09-22 DOI: 10.1016/j.pep.2025.106822

Yong-Hong Nie , Qiang Li , Yan-Ji Lu , Tuo Tang , Xian Hong , Xin-Xin Yue , Zhi-Hui Deng , Tao Wang

{"title":"Expression and purification of C14orf119 and generation of its polyclonal antibody","authors":"Yong-Hong Nie , Qiang Li , Yan-Ji Lu , Tuo Tang , Xian Hong , Xin-Xin Yue , Zhi-Hui Deng , Tao Wang","doi":"10.1016/j.pep.2025.106822","DOIUrl":"10.1016/j.pep.2025.106822","url":null,"abstract":"<div><div>C14orf119 is a functionally uncharacterized mitochondrial protein potentially implicated in ischemic stroke pathogenesis. To enable comprehensive biological studies, we developed and validated a specific polyclonal antibody against this target. Our approach involved prokaryotic expression and chromatographic purification of both His- and GST-tagged C14orf119 in <em>Escherichia coli</em>. The purified His-C14orf119 served as immunogen for rabbit polyclonal antibody production, while the purified GST-C14orf119 enabled subsequent affinity purification of target-specific antibody. Rigorous characterization demonstrated the antibody's exclusive specificity for both exogenous and endogenous C14orf119. Notably, the C14orf119 antibody failed to detect control His-tagged proteins, confirming effective removal of anti-His antibodies during the GST-based affinity purification process. This rigorously validated antibody provides a critical molecular tool for functional characterization of C14orf119, paving the way for mechanistic investigations into its pathophysiological roles.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106822"},"PeriodicalIF":1.2,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145138455","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Expression and purification of Austropuccinia psidii effector proteins in Escherichia coli psidii效应蛋白在大肠杆菌中的表达与纯化。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-09-16 DOI: 10.1016/j.pep.2025.106815

Jovarn V. Sullivan , Michael J. Currie , Vanessa K. Morris , Ashish Sethi , Santosh Panjikar , Grant R. Smith , Claudia-Nicole Meisrimler , Renwick C.J. Dobson

{"title":"Expression and purification of Austropuccinia psidii effector proteins in Escherichia coli","authors":"Jovarn V. Sullivan , Michael J. Currie , Vanessa K. Morris , Ashish Sethi , Santosh Panjikar , Grant R. Smith , Claudia-Nicole Meisrimler , Renwick C.J. Dobson","doi":"10.1016/j.pep.2025.106815","DOIUrl":"10.1016/j.pep.2025.106815","url":null,"abstract":"<div><div>The plant disease myrtle rust is caused by the fungus <em>Austropuccinia psidii</em>. It has led to functional myrtaceous species extinctions in Australia and is a significant threat to other species globally. During infection, <em>A. psidii</em> secretes effector proteins that manipulate the host plant's defences. Numerous putative effectors are encoded in this pathogen's genome, some being expressed early during urediniospore germination and initial invasion of plant tissues. Four putative effector proteins (AP1260, AP5292, AP10948, and AP143) were found to be differentially expressed in the first 24–48 h of infection, suggesting that they play important roles in the infection process. As in other rust fungi, these effector proteins are small and cysteine-rich, often forming disulfide bonds, and their isolation for biophysical characterisation can be challenging. AlphaFold3 models predict that AP1260, AP5292, AP10948, and AP143 form disulfide bonds, while disorder analysis indicates the presence of intrinsically disordered regions. The four putative <em>A. psidii</em> effector proteins were recombinantly produced using SHuffle <em>Escherichia coli</em> cells with an adapted co-expression vector, ‘ApFunCyDisCo’. Three of the effectors were successfully produced, but were insoluble. The fourth effector, AP1260, was successfully produced in the soluble fraction and purified using a four-step process: immobilised metal affinity chromatography, desalting, anion exchange chromatography, and size exclusion chromatography. Circular dichroism spectroscopy revealed that AP1260 has a mainly random coil character, but also has both β-strand and α-helical content. This first successful production and isolation of an <em>A. psidii</em> protein provides a foundation for future investigation of the molecular mechanisms of <em>A. psidii</em> pathogenicity.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106815"},"PeriodicalIF":1.2,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086749","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

Optimized Laccase production from the white rot fungi Pleurotus ostreatus and Trametes versicolor 白腐菌平菇和彩板菌产漆酶的优化。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-09-11 DOI: 10.1016/j.pep.2025.106813

Apoorva Deshmukh , Parnal Sattikar , Aishwarya Sukhatankar , Geetanjali Wakade , Pramod Kumbhar , Phaneeswara-Rao Kommoju

{"title":"Optimized Laccase production from the white rot fungi Pleurotus ostreatus and Trametes versicolor","authors":"Apoorva Deshmukh , Parnal Sattikar , Aishwarya Sukhatankar , Geetanjali Wakade , Pramod Kumbhar , Phaneeswara-Rao Kommoju","doi":"10.1016/j.pep.2025.106813","DOIUrl":"10.1016/j.pep.2025.106813","url":null,"abstract":"<div><div>Laccases are predominantly found in bacteria, fungi, plants, and insects, and they have numerous industrial and biotechnological applications. Laccases are utilized in the pharmaceutical, food, pulp, and paper industries, and their cost-effective production in submerged culture conditions is of significant commercial value. Attempts were made to overexpress three laccase isoforms from <em>Trametes versicolor</em> (TV) in heterologous systems. Recombinant TV laccases were either insoluble in <em>E.coli</em> or poorly expressed in <em>Pichia pastoris.</em> Hence a submerged fermentation process was developed to produce these commercially important laccases from two non-recombinant white rot fungi: <em>Pleurotus ostreatus</em> (PO) and <em>Trametes versicolor</em> (TV). Molasses and corn steep liquor (CSL) were used as carbon and nitrogen sources, respectively, while 2,5-xylidine, sodium lignosulfonate, and copper sulfate were used as inducers, making the entire process economical. Laccase activity reached a maximum of 374,000 U/L (or 374 kU/L) in 20 days. When evaluated at 45 °C, TV laccase outperformed PO laccase in terms of stability, a crucial factor in the delignification of biomass. TV laccase demonstrated superior stability over PO laccase at 45 °C, making it the preferred choice for biomass pre-treatment applications. We demonstrate the use of this laccase in two industrial applications.</div><div>1. Lignocellulose Depolymerization: Treatment of rice straw with <em>Pleurotus ostreatus</em> laccase resulted in visible structural distortion, as observed under a scanning electron microscope.</div><div>2. Industrial Wastewater Treatment: Significant decolorization of indigo carmine and Remazol Brilliant Blue dyes was achieved overnight, with reductions of up to 70 % and 74 %, respectively, when incubated with <em>Trametes versicolor</em> laccase.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106813"},"PeriodicalIF":1.2,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145058529","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