Protein expression and purification最新文献

Human TBC1 domain-containing kinase is a class I multidomain pseudokinase. 人TBC1结构域激酶是一类多结构域假激酶。

IF 1.8 4区生物学

Protein expression and purification Pub Date : 2027-01-01 Epub Date: 2026-08-03 DOI: 10.1016/j.pep.2026.106989

Shiwangi Maurya, Lilly E Cheek, Anthony T Iavarone, Wen Zhu

引用次数: 0

Biochemical characterization of recombinant L-threonine 3-dehydrogenase from Akkermansia muciniphila. 嗜粘杆菌重组l -苏氨酸3-脱氢酶的生化表征。

IF 1.8 4区生物学

Protein expression and purification Pub Date : 2026-10-16 DOI: 10.1016/j.pep.2026.107028

Xue Dong, Jiatong Zhao, Lanlan Mei, Xueru Liu

引用次数: 0

Simplified Chromatography-based Purification approach with utilizing Tangential Flow Filtration for Colorectal Cancer Biomarker: Carcinoembryonic Antigen. 基于切向流过滤的简化色谱纯化大肠癌生物标志物：癌胚抗原。

IF 1.8 4区生物学

Protein expression and purification Pub Date : 2026-10-16 DOI: 10.1016/j.pep.2026.107027

Arti M Rane, Shamkant B Badgujar, Kunal K Shukla, Rohini S Daftary, Shraddha V Prabhu, Aruni Wilson Santosh Kumar, Vinoth P Gunasekaran

{"title":"Simplified Chromatography-based Purification approach with utilizing Tangential Flow Filtration for Colorectal Cancer Biomarker: Carcinoembryonic Antigen.","authors":"Arti M Rane, Shamkant B Badgujar, Kunal K Shukla, Rohini S Daftary, Shraddha V Prabhu, Aruni Wilson Santosh Kumar, Vinoth P Gunasekaran","doi":"10.1016/j.pep.2026.107027","DOIUrl":"https://doi.org/10.1016/j.pep.2026.107027","url":null,"abstract":"<p><p>Carcinoembryonic antigen (CEA) is a clinically important biomarker for cancer diagnosis and prognosis, particularly in colorectal cancer. This investigation introduces a simplified approach for the purification of native CEA antigen from the ascitic fluid of patients with colorectal cancer. Moreover, the technique allows the characterization of CEA though ensuring its structural and functional integrity is preserved. The CEA purification was achieved using perchloric acid precipitation, tangential flow filtration and sequential chromatography. The isolated CEA showed a relative molecular mass of ∼200 kDa and was analyzed using SDS-PAGE, size-exclusion FPLC and immunoblotting, confirming high purity comparable to reference form of native CEA protein. Peptide mass fingerprinting of trypsin-digested and deglycosylated CEA antigen identified it as CEACAM5_HUMAN (Uniprot ID: P06731). The proposed method demonstrates the adaptability for larger volume and practical approach for production of native form of CEA protein, also potential utility for future diagnostic applications, clinical research and bioanalytical studies.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"107027"},"PeriodicalIF":1.8,"publicationDate":"2026-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149207864","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

Development of an intein-based bioprocessing platform for recombinant production of a potent antimicrobial peptide: Lasioglossin-III. 基于蛋白的重组生产高效抗菌肽的生物加工平台：激光舌蛋白- iii。

IF 1.8 4区生物学

Protein expression and purification Pub Date : 2026-10-09 DOI: 10.1016/j.pep.2026.107025

Atul Kumar, Manju Nagar Galodiya, Sankar Chakma, Anita Dutt Konar, Anindya Basu

{"title":"Development of an intein-based bioprocessing platform for recombinant production of a potent antimicrobial peptide: Lasioglossin-III.","authors":"Atul Kumar, Manju Nagar Galodiya, Sankar Chakma, Anita Dutt Konar, Anindya Basu","doi":"10.1016/j.pep.2026.107025","DOIUrl":"10.1016/j.pep.2026.107025","url":null,"abstract":"<p><p>The rapid emergence of antimicrobial resistance has intensified the demand for novel anti-infective agents, positioning antimicrobial peptides (AMPs) as promising therapeutic alternatives. However, large scale production of AMPs remains challenging due to their small size, cationic nature, host toxicity, and high cost of chemical synthesis. In this study, we report an efficient and economically viable intein-mediated recombinant bioprocess for the production of the short antimicrobial peptide Lasioglossin-III (LL-III) in its C-terminally amidated form using Escherichia coli-based expression platform. A codon-optimized LL-III gene was fused to the Mxe GyrA intein along with a chitin binding domain (CBD) using the pTWIN1 expression system, enabling stable expression, affinity purification, and controlled auto-cleavage. The fusion protein was expressed in soluble form at low temperature and purified using chitin resin under optimized binding conditions. Notably, the chitin resin retained its binding capacity across multiple reuse cycles, improving the overall process-economics; auto-cleavage under mild reducing conditions released the mature peptide, which was confirmed by RP-HPLC and LC/MS to be identical to chemically synthesized standard peptide. The optimized process yielded ∼4.4 mg L<sup>-1</sup> of recombinant peptide leading to significant reduction in production costs at the current cleavage efficiency. Overall, this study establishes a scalable, environmentally sustainable, and cost-effective bioprocessing platform not only for short AMPs, but also for other peptide-based therapeutics as well.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"107025"},"PeriodicalIF":1.8,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149149494","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

Fine-tuning of leaky expression delivers soluble and functional recombinant alphainterferon in the cytoplasm of Escherichia coli 在大肠杆菌细胞质中，对泄漏表达的微调可提供可溶性和功能性的重组α干扰素

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-01-28 DOI: 10.1016/j.pep.2026.106895

Rodrigo Martins Bretas , Marcus Vinícius Alves da Silva , Sophie Yvette Leclercq , Diana Paola Gómez-Mendoza , Armando da Silva Cunha Jr. , Luciana Maria Silva Lopes

{"title":"Fine-tuning of leaky expression delivers soluble and functional recombinant alphainterferon in the cytoplasm of Escherichia coli","authors":"Rodrigo Martins Bretas , Marcus Vinícius Alves da Silva , Sophie Yvette Leclercq , Diana Paola Gómez-Mendoza , Armando da Silva Cunha Jr. , Luciana Maria Silva Lopes","doi":"10.1016/j.pep.2026.106895","DOIUrl":"10.1016/j.pep.2026.106895","url":null,"abstract":"<div><div>Alphainterferons are cytokines that became famous in the treatment of certain infections and tumors due to their antiviral, antiproliferative, and immunomodulatory activities. While the production of these small proteins in <em>Escherichia coli</em> is well established, high-level overexpression typically results in the formation of insoluble and inactive inclusion bodies that need to be processed through costly and lengthy steps. Here we describe an optimized, inducer-free expression system using <em>E. coli</em> BL21 (DE3) and a pET-based vector with the human IFN-α2a gene. By strategically transitioning the culture from high-temperature biomass accumulation (37 °C) to mild conditions (16 °C) during the early-midlog phase, we leveraged basal expression to favor folding over aggregation. This approach achieved a cytoplasmic solubility of up to 75 %. The protein was recovered using a streamlined one- or two-step ion-exchange chromatography protocol, resulting in maximum purified yields of 23.5 mg/L. Characterization tests confirmed the protein's identity, high purity, <em>in vitro</em> activity, and correct disulfide bonding pattern. This method provides a cost-effective alternative for the production of soluble alphainterferon by eliminating the need for chemical inducers and complex refolding processes.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106895"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146079640","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

Scalable production and characterization of recombinant pneumococcal histidine triad protein D in Escherichia coli for vaccine development 重组肺炎球菌组氨酸三联体蛋白D在大肠杆菌中的规模化生产和特性研究

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-01-13 DOI: 10.1016/j.pep.2026.106889

Prashant Jadhav , Hitesh Malviya , Jyoti Gokhale

{"title":"Scalable production and characterization of recombinant pneumococcal histidine triad protein D in Escherichia coli for vaccine development","authors":"Prashant Jadhav , Hitesh Malviya , Jyoti Gokhale","doi":"10.1016/j.pep.2026.106889","DOIUrl":"10.1016/j.pep.2026.106889","url":null,"abstract":"<div><div><em>Streptococcus pneumoniae</em> causes 1.5 million deaths annually, primarily in low-income countries. Pneumococcal histidine triad protein D (PhtD), a conserved 92–110 kDa surface protein, is a promising serotype-independent vaccine candidate due to its roles in adherence, zinc homeostasis, and complement evasion. This study optimized scalable production of His-tagged PhtD in <em>Escherichia coli</em> BL21(DE3). The PhtD gene (serotype 2, strain D39) was cloned into pET30a(+), and expression was optimized using a full factorial design of experiments, achieving up to 19.8 mg/L soluble PhtD (>90 % purity) with 0.1 mM IPTG at OD<sub>590</sub> = 0.2 for 2 h. Lysozyme treatment (0.3 mg/mL) increased yield to 22 mg/L, a 120 % improvement over the control (10 mg/L). Purification involved sonication, ultrafiltration, and IMAC (80–100 mM imidazole wash, 200–300 mM elution), with 1 mM PMSF preventing ∼30 % yield loss from proteolysis. PhtD's identity (105–110 kDa), secondary structure (15.5 % helix, 30.4 % sheet), and thermal stability (Tm ∼102 °C) were confirmed by SDS-PAGE, Western blotting, FTIR, circular dichroism, NMR, and DSC. This optimized, statistically guided process delivers the highest reported yield of soluble full-length PhtD in cytoplasmic <em>E. coli</em> with documented native-like folding and exceptional stability (Tm = 102 °C), providing a robust and scalable platform for serotype-independent pneumococcal vaccine development.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106889"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145981457","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

Detection of human PD-1 on T-cells using a fusion protein consisting of an anti-PD-1 single chain variable fragment linked to superfolder GFP 使用与超级文件夹gfp连接的抗pd-1单链可变片段组成的融合蛋白检测t细胞上的人pd-1。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2025-12-28 DOI: 10.1016/j.pep.2025.106876

Jong Shin , David Sanford , Alex A. Bohm , William Bachovchin , Peter A. Bullock

{"title":"Detection of human PD-1 on T-cells using a fusion protein consisting of an anti-PD-1 single chain variable fragment linked to superfolder GFP","authors":"Jong Shin , David Sanford , Alex A. Bohm , William Bachovchin , Peter A. Bullock","doi":"10.1016/j.pep.2025.106876","DOIUrl":"10.1016/j.pep.2025.106876","url":null,"abstract":"<div><div>Monoclonal antibodies that disrupt the interaction between T-cell derived PD-1 and immunosuppressive ligands, such as PD-L1, have dramatically improved approaches to cancer therapy. One such antibody is termed Nivolumab (trade name Opdivo) that selectively targets PD-1. We previously described a Nivolumab-based anti-PD-1 single chain variable fragment (scFv), termed the anti-PD-1 double mutant (dm) scFv, that tightly binds to PD-1 and blocks the interaction between PD-1 expressed on T-cells and PD-L1 on CHO cells. Extending these experiments, we now report a fluorescent reporter formed by the fusion of the anti-PD-1dm with superfolder (sf) GFP. Immunofluorescence and flow cytometry studies established that the anti-PD-1dm-sfGFP fusion protein can be used to detect PD-1 on the surface of PD-1 expressing Jurkat cells. Furthermore, using a humanized mouse strain that expresses human PD-1 and PD-L1, we report that when combined with an anti-CD3 antibody, the anti-PD-1dm-sfGFP can be used to identify PD-1 expressing T-cells within mouse tumor biopsies. Thus, we have developed a reagent for the quantitative determination of activated T-cells in tumor biopsies. Finally, we report that an additional fluorescent probe for the detection of PD-1 <em><u>in vivo</u></em> can be generated by linking, via maleimide chemistry, a derivative of the anti-PD-1dm to the near-infrared dye IR800.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106876"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145865191","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

Removal of polysorbates degradation-related impurities by depth filter screening: A case study 深度过滤筛选去除聚山梨酯降解相关杂质：一个案例研究。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-01-30 DOI: 10.1016/j.pep.2026.106893

Chunxiao Xuan , Yangguang Xu , Xinang Cui, Tengfei Ma, Fengjuan Lv

引用次数: 0

Bioinformatics analysis and preparation of polyclonal antibodies against the extracellular region of the Langya Virus attachment protein 狼牙病毒附着蛋白胞外区多克隆抗体的制备及生物信息学分析。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-01-22 DOI: 10.1016/j.pep.2026.106888

Shuxing Qiu , Xing Yin , Jinjiao He , Jiayou Zhang , Jinjing Guo , Yonghua Qi , Feifan Liu

{"title":"Bioinformatics analysis and preparation of polyclonal antibodies against the extracellular region of the Langya Virus attachment protein","authors":"Shuxing Qiu , Xing Yin , Jinjiao He , Jiayou Zhang , Jinjing Guo , Yonghua Qi , Feifan Liu","doi":"10.1016/j.pep.2026.106888","DOIUrl":"10.1016/j.pep.2026.106888","url":null,"abstract":"<div><div>The Langya virus attachment glycoprotein (LayV-G) is a major surface glycoprotein critical for host cell entry, membrane fusion, and the induction of neutralizing antibody production. A highly specific, reliable, and cost-effective anti-LayV-G antibody is urgently needed to comprehensively assess its multifunctional role in the viral life cycle. Here, we report the first successful production of LayV-G protein and corresponding polyclonal antibodies (pAbs) using a prokaryotic expression system. In this study, the extracellular domain of LayV-G containing strong antigenic epitopes was predicted using bioinformatics approaches and selected for recombinant antigen production, followed by codon optimization and synthesis of the candidate protein-encoding gene. We then constructed the truncated attachment glycoprotein (tG) with a C-terminal histidine tag (His-tag), (pET-22b-tG/his) recombinant plasmid and expressed the protein in <em>E. coli</em> BL21 (DE3) competent cells. SDS–PAGE and subsequent tandem mass spectrometry revealed the molecular weight of the tG/his recombinant protein to be ∼64 kDa (kD). Subsequently, the targeted protein was used to immunize New Zealand white rabbits and generate pAbs. The specificity and titer of the pAbs were determined by Western blotting, indirect fluorescence assay (IFA), and indirect ELISA. The antibody exhibited high specificity for tG/his in prokaryotic cells (Western blotting), and LayV-G expressed by the recombinant baculovirus system (IFA). In conclusion, the LayV-G protein and pAbs targeting it lays the foundation for further investigations into LayV-G structure and function, pathogenic LayV molecular mechanisms, and rapid LayV detection methods.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106888"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146044040","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

Robust aggregate separation by Sartobind Rapid A Protein A membrane sarbinding Rapid A Protein A膜的稳健聚集体分离。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-01-24 DOI: 10.1016/j.pep.2026.106890

Gaoya Yuan, Meng Qu, Yifeng Li

{"title":"Robust aggregate separation by Sartobind Rapid A Protein A membrane","authors":"Gaoya Yuan, Meng Qu, Yifeng Li","doi":"10.1016/j.pep.2026.106890","DOIUrl":"10.1016/j.pep.2026.106890","url":null,"abstract":"<div><div>Protein A resins are the most widely used affinity media for product capture in antibody and Fc-fusion protein purification. However, they generally lack the capability to separate monomers from co-binding aggregates. Recently, Protein A membrane has emerged as a promising alternative to resin-based Protein A column. We previously demonstrated that Sartobind Rapid A, a Protein A membrane from Sartorius, shows much stronger aggregate separation capability than resin-based Protein A columns. In the current work, using an aggregate-rich culture harvest as feed material, we evaluated three mobile phase additives (i.e., NaCl, CaCl<sub>2</sub> and Arg·HCl) for their effects on monomer-aggregate separation by Sartobind Rapid A membrane. After identifying the conditions that provided the best resolution, we challenged Sartobind Rapid A membrane with four artificial samples containing different ratios (i.e., 15%, 30%, 45% and 60%) of aggregates. Analysis of the main elution peak portion by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) suggested that this Protein A membrane effectively removed most aggregates in all artificial samples regardless of the percentage of aggregates in them. Thus, the current study shows that aggregate separation by Sartobind Rapid A membrane is not only effective but also highly robust, which makes Protein A membrane a more attractive choice than Protein A column for product capture, especially for cases where the culture harvest contains high amounts of aggregates.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106890"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146053469","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