Protein expression and purification最新文献

{"title":"Recombinant expression of crotoxin and comparison to native crotoxin as immunogens for anti-crotalid antivenoms","authors":"Miguel Angel Mejía Sánchez ,&nbsp;Samuel Cardoso Arenas ,&nbsp;Ricardo Miranda Blancas ,&nbsp;Herlinda Clement ,&nbsp;Ligia Luz Corrales García ,&nbsp;Adrián Marcelo Franco-Vásquez ,&nbsp;Ivan Arenas ,&nbsp;Alejandro Carbajal Saucedo ,&nbsp;Gerardo Corzo","doi":"10.1016/j.pep.2025.106871","DOIUrl":"10.1016/j.pep.2025.106871","url":null,"abstract":"<div><div>The crotoxin complex is identified as the primary lethal toxin in various species of rattlesnakes. It comprises two subunits: CtxA, a non-toxic protein, and CtxB, a minimally toxic enzymatic phospholipase A₂. These components exert neurotoxic effects by disrupting neuromuscular transmission, specifically by inhibiting the release of acetylcholine, thereby causing flaccid paralysis of the muscles. Due to crotoxin's high lethality, variable presence, and proportion within crotalid venoms, considerable interest has been directed toward its inhibition. In this context, the heterologous expression of a recombinant fusion protein designated as rCtxBA, which incorporates the amino acid sequences of both CtxA and CtxB subunits of crotoxin, has been documented. The rCtxBA protein was recombinantly expressed, recovered, and subsequently purified. New Zealand rabbits were immunized with the recombinants rCtxBA, rCTxB, and the native CtxBA. ELISA and Western blot analyses revealed that the anti-crotoxin antibodies specifically recognized venoms containing crotoxin by binding to both conformational and linear epitopes of crotoxin-like or related phospholipases. <em>In vivo</em> neutralization assays performed in mice demonstrated efficacy of the anti-rCtxBA antibodies against venoms from <em>Crotalus mictlantecuhtli, C. scutulatus salvini</em>, and <em>C. tzabcan—</em>species known to contain crotoxin—with ED₅₀ values of 2.8, 7.8, and 2.5 mg/3LD₅₀, respectively. Conversely, no neutralization effect was observed against the venom of <em>C. molossus nigrescens</em>, a species lacking crotoxin in its venom profile. These findings substantiate that native crotoxin, present in whole crotalid venoms, can be neutralized using recombinant crotalid antigens, thereby facilitating the development of effective neutralizing antibodies.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106871"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145775233","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}

{"title":"Recombinant expression of antimicrobial peptide AMP-PD as tandem octamer and its antibacterial activity in vitro","authors":"Xingmiao Lu ,&nbsp;Yuan Sun ,&nbsp;Ziyi Wang ,&nbsp;Xiaoke Jin ,&nbsp;Jiayu Zhang ,&nbsp;Fuping Lu ,&nbsp;Wen-Chao Yang ,&nbsp;Fufeng Liu","doi":"10.1016/j.pep.2025.106839","DOIUrl":"10.1016/j.pep.2025.106839","url":null,"abstract":"<div><div>The widespread misuse of antibiotics has led to increased bacterial resistance, posing a severe threat to public health. Antimicrobial peptides (AMPs) have emerged as promising alternatives due to their low resistance and broad-spectrum antibacterial activity. However, the low heterologous expression efficiency of AMPs has hindered their large-scale application. Herein, a novel tandemly gene duplication expression system of AMP was developed with recombinant plasmid His₆-AMP-PD₈ that containing eight OM19R repeats and tandemly linked by aspartic acid (D) and proline (P), using AMP-PD (PVDKPPYLPRPRPIRRPGGRD) as a model. Soluble expression of the fusion protein was successfully achieved in <em>E</em>. <em>coli</em> BL21(DE3). The expression efficiency of the fusion protein, including induction temperature, induction time, and IPTG concentration, was optimized to enhance the yield of the recombinant protein. About 32 mg/L the recombinant protein was achieved under the optimal condition, 35 °C, 0.7 mmol/L IPTG and 10 h of induction time. Then, 16 mg/L AMP-PD was obtained after cleavage with 50 % formic acid at 55 °C for 24 h and isolated using 3 kDa ultrafiltration device. Finally, the AMP-PD exhibited considerable antibacterial activity with 12.59 mm of inhibition circle diameter, and the MIC and MBC for <em>E. coli</em> ATCC25922 were 45.75 μg/mL and 62.53 μg/mL, respectively. This study successfully realized the soluble expression system of AMP-PD, laying the foundation for its industrial production and general application for AMP biosynthesis.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106839"},"PeriodicalIF":1.2,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145368634","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}

{"title":"Study on the hole and knob related impurities in the purification of bispecific antibodies","authors":"Huanlan Chen,&nbsp;Naiqi Ji,&nbsp;Maozhi Zhou,&nbsp;Yingyue Bu","doi":"10.1016/j.pep.2025.106836","DOIUrl":"10.1016/j.pep.2025.106836","url":null,"abstract":"<div><div>The Knob-into-Hole (KiH) design is an effective strategy to promote the heterodimerization of heavy chains. In this research, through the integration of advanced analytical techniques, we made a critical finding regarding heterodimer formation. When mixing materials derived from different clones containing hole-hole homodimers and knob-knob homodimers, the resulting structures initially appear to form the desired knob-into-hole (KiH) configuration. However, detailed characterization by SDS-Caliper NR analysis revealed these were not genuine KiH heterodimers. While this phenomenon presents an optimal apparent outcome, it has significant impact on the optimization of process in heterodimer production. Additionally, a comprehensive assessment was carried out on the efficacy of cation exchange chromatography (CEX) in removing homodimers. This study presents a case for the optimization of the CEX process, utilizing bispecific - antibody materials sourced from different clonal bioreactors. By adjusting the operating pH of CEX, the knob-related impurities and hole-related impurities were reduced to below the detection limit, as shown by iCIEF analysis. Meanwhile, SEC-UPLC analysis indicated that purity increased to 99 %, and SDS-Caliper NR results showed that the percentage of functional KiH improved from 85 % in the input to 97 % after elution. These findings demonstrate that the CEX process contributes to an increase in the proportion of functional KiH.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106836"},"PeriodicalIF":1.2,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145337454","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}

{"title":"Catalytically active hCELA3B is a natively-folded monomer and is N-glycosylated","authors":"Prince Kumar ,&nbsp;Prabhakar Babele ,&nbsp;Rishav Madhukalya ,&nbsp;Abhishek Goswami ,&nbsp;Khadijah Ameen Khan ,&nbsp;Rohit Gupta ,&nbsp;Anica Dadwal ,&nbsp;Rajesh Kumar ,&nbsp;Dilip Kumar ,&nbsp;Pramod Kumar Garg ,&nbsp;Supratik Das","doi":"10.1016/j.pep.2025.106840","DOIUrl":"10.1016/j.pep.2025.106840","url":null,"abstract":"<div><div>Chymotrypsin-like elastases CELA3A and CELA3B constitute a sub-family of serine proteases that hydrolyze proteins such as elastin. They are secreted from the pancreas as zymogens, are activated catalytically after cleavage by trypsin, and have digestive function in the intestine. A decrease in these enzymes, due to pancreatitis, can lead to pancreatic exocrine insufficiency (PEI). However, the properties and structures of human CELA3A and CELA3B remain to be determined. To address this, we developed a method to express and purify pro-hCELA3A and pro-hCELA3B from mammalian suspension cultures. We report that the protein can be rapidly purified from the supernatant of expression cultures to apparent homogeneity. The proteins are natively folded and enzymatically active. Only pro-hCELA3B forms homogeneous monomers making it suitable for further structural studies. Pro-hCELA3B is N-glycosylated.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106840"},"PeriodicalIF":1.2,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145445514","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}

{"title":"Development and expression of a factor X-derived recombinant protein for ELISA-based detection of Emicizumab","authors":"Sina Tavakoli ,&nbsp;Shohreh Zare Karizi ,&nbsp;Jafar Amani","doi":"10.1016/j.pep.2025.106829","DOIUrl":"10.1016/j.pep.2025.106829","url":null,"abstract":"<div><div>Emicizumab is a therapeutic antibody used in the management of Hemophilia A. It mimics the function of coagulation Factor VIII by bridging activated Factor IX (FXa) and Factor X (FX), thereby facilitating the activation of FX and promoting blood coagulation. Due to its prolonged half-life in patient plasma following administration, accurate detection of Emicizumab is clinically important. Enzyme-linked immunosorbent assay (ELISA) is a valuable method for monitoring Emicizumab levels, but it requires proteins that bind with high affinity to the antibody.</div><div>In this study, we engineered and expressed a recombinant fragment of Factor X containing the EGF-like 2 domain, designed to bind Emicizumab. A gene fragment encoding the EGF-like 2 domain of Factor X, previously identified as an Emicizumab-binding region, was codon-optimized and cloned into pUC57, then subcloned into the pET28a expression vector. <em>Escherichia coli</em> DH5α and <em>E. coli</em> Shuffle T7 (DE3) strains were used as cloning and expression hosts, respectively. The recombinant protein was purified and verified using 10 % SDS-PAGE, Bradford assay, and Western blot. Following expression, the recombinant protein was purified and quantified, with concentrations of 119 μg/mL for native and 81 μg/mL for denatured protein. These values correspond to total recoveries of 255 μg and 65 μg, respectively, from 50 mL cultures. These findings validate the use of this bacterially expressed FX-derived protein as a candidate component for incorporation into ELISA-based platforms aimed at Emicizumab detection.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106829"},"PeriodicalIF":1.2,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145308822","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}

{"title":"The long chain fatty acid-CoA ligase VraA plays a regulatory role in vancomycin resistance in Staphylococcus aureus","authors":"Dafeng Liu ,&nbsp;Na Li","doi":"10.1016/j.pep.2025.106838","DOIUrl":"10.1016/j.pep.2025.106838","url":null,"abstract":"<div><div>The pathogen <em>Staphylococcus aureus</em> threatens clinical and public health. Vancomycin, a former last-resort treatment, has waning efficacy against resistant strains (VISA) that alter their cell envelope using enzymes like VraA. However, the specific functional mechanism of VraA in VISA is still not understood. Here, we successfully obtained monomeric VraA and found that oleate is the optimal substrate for VraA. VraA was characterized as a peripheral membrane protein with a hydrodynamic radius of 5.7 ± 0.3 nm. Disruption of <em>VraA</em> significantly increased <em>S. aureus</em> susceptibility to vancomycin, as demonstrated by minimum inhibitory concentration (MIC) assays and RT-qPCR. The structural model of VraA predicted using AlphaFold2 was refined via ModRefiner, and active site residues were identified. Our findings underscore the pivotal role of VraA in mediating vancomycin resistance in <em>S. aureus</em>, providing valuable insights for developing targeted therapies against this pathogen.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106838"},"PeriodicalIF":1.2,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145355741","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}

{"title":"Expression, purification and functional validation of a cancer-associated isoform of the HBx protein from human hepatitis B virus","authors":"Alexis Clavier ,&nbsp;Santiago Gómez-Evain ,&nbsp;Toshinobu Shida ,&nbsp;Rubaba R. Abanti ,&nbsp;Franziska Hammerstein ,&nbsp;Pavel Kielkowski ,&nbsp;Mila Leuthold ,&nbsp;Anne K. Schütz","doi":"10.1016/j.pep.2025.106842","DOIUrl":"10.1016/j.pep.2025.106842","url":null,"abstract":"<div><div>The human hepatitis B virus (HBV) causes hepatitis B, a liver infection that can be acute or chronic. HBV encodes four proteins, among which the X protein (HBx) plays a critical role in viral replication. During chronic HBV infection, in which the viral DNA is integrated into the host genome, the HBx_1-120 isoform, comprising the N-terminal 120 residues, is highly expressed. Here, we describe a protocol for the recombinant overexpression and purification of untagged HBx_1-120 from bacterial cells. The procedure is compatible with stable isotope labelling in minimal media. Following cell lysis, HBx_1-120 was recovered from inclusion bodies (IBs), solubilized in urea, and purified by ion-exchange (IEX) and size-exclusion chromatography (SEC). The purified protein was extensively characterized, including by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Functionality was confirmed by a pulldown assay with a known interacting partner, Spindlin1. This protocol provides a robust framework to obtain untagged HBx_1-120 for structural and functional <em>in vitro</em> studies.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106842"},"PeriodicalIF":1.2,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145452644","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}

{"title":"Protein production in transgenic chickens mediated through primordial germ cells and embryonic stem cells","authors":"Mengran Fang ,&nbsp;Xinyue Chen ,&nbsp;Yuan Liu,&nbsp;Hui Yuan","doi":"10.1016/j.pep.2025.106828","DOIUrl":"10.1016/j.pep.2025.106828","url":null,"abstract":"<div><div>The inherent advantages of transgenic chickens, combined with the increasing demand for therapeutic proteins, are driving significant progress in transgenic chicken bioreactors. Primordial germ cells (PGCs) and embryonic stem cells (ESCs) are two pivotal carrier cells in the production of transgenic chicken bioreactors. Transgenic chickens have successfully produced therapeutic proteins using genetically modified and transferred PGCs. Meanwhile, considerable progress has been made in expressing therapeutic proteins using exogenous gene integration mediated by ESCs. Both carrier cells facilitate the efficient process of transgenic chickens. Here, we reviewed the characteristics, current applications, comparisons, and future directions of these two carrier cells, providing theoretical foundations and practical guidance for advancing development in the fields of chicken bioreactors.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106828"},"PeriodicalIF":1.2,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145337522","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}

{"title":"Effect of culture media and fermentation process on the refolding and purification of rh-GCSF","authors":"Somayeh Abolghasemi ,&nbsp;Fatemeh Poureini ,&nbsp;Valiollah Babaeipour ,&nbsp;Faezeh Farji ,&nbsp;Mohammad Reza Mofid","doi":"10.1016/j.pep.2025.106837","DOIUrl":"10.1016/j.pep.2025.106837","url":null,"abstract":"<div><div>Human granulocyte-colony stimulating factor (h-GCSF) is used to mitigate neutropenia caused by chemotherapy. <em>E. coli</em> is the most common host for h-GCSF production due to its high yield and versatile culture strategies. However, overproduction of h-GCSF in <em>E. coli</em> often results in the formation of inclusion bodies (IBs) in the cytoplasm. The efficiency of refolding and purifying recombinant h-GCSF (rh-GCSF) produced as IBs depends on the expression strategy, induction, and cell growth conditions. This study investigated how different culture media and fermentation processes affect the refolding of IBs and the purification of rh-GCSF. The purified samples were compared to Neupogen® and PDgrastim reference standards using Western blotting, size exclusion chromatography, and reverse-phase high-performance liquid chromatography. The analysis confirmed that all proteins were correctly refolded and exhibited a purity exceeding 90 % after purification. The highest protein recovery percentage and purity of rh-GCSF with the lowest endotoxin content was achieved using M9 media in fed-batch culture.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106837"},"PeriodicalIF":1.2,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145346688","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}

{"title":"Optimized Laccase production from the white rot fungi Pleurotus ostreatus and Trametes versicolor","authors":"Apoorva Deshmukh ,&nbsp;Parnal Sattikar ,&nbsp;Aishwarya Sukhatankar ,&nbsp;Geetanjali Wakade ,&nbsp;Pramod Kumbhar ,&nbsp;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":"2026-01-01","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}