Protein expression and purification最新文献_第9页

Characterization of a highly thermostable recombinant xylanase from Anoxybacillus ayderensis 来自 Anoxybacillus ayderensis 的高热稳定性重组木聚糖酶的表征

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-04-01 DOI: 10.1016/j.pep.2024.106478

Zuleyha Akpinar , Hakan Karaoglu

{"title":"Characterization of a highly thermostable recombinant xylanase from Anoxybacillus ayderensis","authors":"Zuleyha Akpinar , Hakan Karaoglu","doi":"10.1016/j.pep.2024.106478","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106478","url":null,"abstract":"<div>Xylanases are the main enzymes to hydrolyze xylan, the major hemicellulose found in lignocellulose. Xylanases also have a wide range of industrial applications. Therefore, the discovery of new xylanases has the potential to enhance efficiency and sustainability in many industries. Here, we report a xylanase with thermophilic character and superior biochemical properties for industrial use. The new xylanase is discovered in Anoxybacillus ayderensis as an intracellular xylanase (AAyXYN329) and recombinantly produced. While AAyXYN329 shows significant activity over a wide pH and temperature range, optimum activity conditions were determined as pH 6.5 and 65 °C. The half-life of the enzyme was calculated as 72 h at 65 °C. The enzyme did not lose activity between pH 6.0–9.0 at +4 °C for 75 days. Km, kcat and kcat/Km values of AAyXYN329 were calculated as 4.09824 ± 0.2245 μg/μL, 96.75 1/sec, and 23.61/L/g.s −1, respectively. In conclusion, the xylanase of A. ayderensis has an excellent potential to be utilized in many industrial processes.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140341951","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

Synthesis of BLF1-containing trimethyl chitosan nanoparticles and evaluation of its immunogenicity and protection in syrian mice by oral and subcutaneous injections 合成含 BLF1 的三甲基壳聚糖纳米颗粒，并通过口服和皮下注射评估其在叙利亚小鼠中的免疫原性和保护作用。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-03-30 DOI: 10.1016/j.pep.2024.106462

Ayoub fazeli, Hosein Honari, Davoud Sadeghi, Hamid Bakhtiari

{"title":"Synthesis of BLF1-containing trimethyl chitosan nanoparticles and evaluation of its immunogenicity and protection in syrian mice by oral and subcutaneous injections","authors":"Ayoub fazeli, Hosein Honari, Davoud Sadeghi, Hamid Bakhtiari","doi":"10.1016/j.pep.2024.106462","DOIUrl":"10.1016/j.pep.2024.106462","url":null,"abstract":"<div>The bacterium Burkholderia pseudomallei is the cause of melioidosis infectious disease. In this bacterium, the BLF1 protein wide inhibits the synthesis of proteins in human cells. This disease is reported to cause a death rate of 40% in some parts of the world. Currently, no effective vaccine is available against this bacterial infection. In this study, therefore, a Nano vaccine was synthesized based on the trimethyl chitosan (TMC) polymer containing the BLF1 recombinant protein, and its immunogenicity and protection in Syrian mice were evaluated by oral and subcutaneous injections. The BLF1 recombinant protein expression was induced in Escherichia coli Bl21 (DE3) and purified by the affinity chromatography technique. Recombinant protein-containing nanoparticles (NPs) were then synthesized by the ionotropic gelation method. After oral and subcutaneous injections, antibody titration was assessed by the indirect ELISA assay. Finally, murine groups were challenged using the BLF1 toxin. The results indicated that the immune system showed more antibody titration in subcutaneous injection than in the oral form. However, the results were reversed in the challenge results, and the survival rate was more significant in the oral injection.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140331977","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-yield and cost-effective biosynthesis process for producing antimicrobial peptide AA139 生产抗菌肽 AA139 的高产、低成本生物合成工艺。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-03-27 DOI: 10.1016/j.pep.2024.106475

Ying Zhang , Yapeng Wang , Jianguang Lu , Zongqing Huang , Haoju Hua , Yanan Li , Jun Xu , Jun Feng

{"title":"High-yield and cost-effective biosynthesis process for producing antimicrobial peptide AA139","authors":"Ying Zhang , Yapeng Wang , Jianguang Lu , Zongqing Huang , Haoju Hua , Yanan Li , Jun Xu , Jun Feng","doi":"10.1016/j.pep.2024.106475","DOIUrl":"10.1016/j.pep.2024.106475","url":null,"abstract":"<div>AA139, a variant of natural antimicrobial peptide (AMP) arenicin-3, displayed potent activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria. Nevertheless, there were currently few reports on the bioprocess of AA139, and the yields were less than 5 mg/L. Additionally, it was difficult and expensive to prepare AA139 through chemical synthesis due to its complex structure. These factors have impeded the further research and following clinical application of AA139. Here, we reported a bioprocess for the preparation of AA139, which was expressed in Escherichia coli (E. coli) BL21 (DE3) intracellularly in a soluble form via SUMO (small ubiquitin-related modifier) fusion technology. Then, recombinant AA139 (rAA139, refer to AA139 obtained by recombinant expression in this study) was obtained through the simplified downstream process, which was rationally designed in accordance with the physicochemical characteristics. Subsequently, the expression level of the interest protein was increased by 54% after optimization of high cell density fermentation (HCDF). Finally, we obtained a yield of 56 mg of rAA139 from 1 L culture with a purity of 98%, which represented the highest reported yield of AA139 to date. Furthermore, various characterizations were conducted to confirm the molecular mass, disulfide bonds, and antimicrobial activity of rAA139.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140327074","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A two-step method preparation of semaglutide through solid-phase synthesis and inclusion body expression 通过固相合成和包涵体表达两步法制备塞马鲁肽

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-03-23 DOI: 10.1016/j.pep.2024.106477

Dezheng Peng , Yang Li , Linlin Si , Bo Zhu , Peng Wu , Yibang Li , Dongfang Tang , Yu Liu , Yunxiao Zhang

{"title":"A two-step method preparation of semaglutide through solid-phase synthesis and inclusion body expression","authors":"Dezheng Peng , Yang Li , Linlin Si , Bo Zhu , Peng Wu , Yibang Li , Dongfang Tang , Yu Liu , Yunxiao Zhang","doi":"10.1016/j.pep.2024.106477","DOIUrl":"10.1016/j.pep.2024.106477","url":null,"abstract":"<div>Semaglutide is currently the most promising antidiabetic drug, especially for the treatment of type 2 diabetes mellitus, due to its excellent efficacy in glycemic control and weight loss. However, the production of semaglutide remains high cost, and high yield, low cost, and high purity still remains a challenge. Herein, we reported a convenient and high-yield strategy for the preparation of semaglutide through fragmented condensation coupling, involving solid-phase peptide synthesis of tetrapeptide and on-column refolding and on-column enzyme cleavage based inclusion body expression of Lys26Arg34GLP-1 (11–37) with fused protein tags in an X-Y-D4K-G pattern. The optimized N-terminal protein tag significantly boosts inclusion body expression level, while on-column refolding and on-column enzyme cleavage avoid precipitation, enhancing efficiency and yield together with one-step purification. The successful preparation of semaglutide is expected to achieve large-scale industrial production with low cost, high yield and high purity.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140282895","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

Purification, crystallization and preliminary crystallographic analysis of Chlamydophila pneumoniae AP endonuclease IV 肺炎衣原体 AP 内切酶 IV 的纯化、结晶和初步晶体学分析。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-03-22 DOI: 10.1016/j.pep.2024.106476

Yitong Zhang , Yangjie Ren , Ben Wang, Shiyang Guo, Siqi Wang, Jinglin Jin, Lihong Yang, Wei Gao

{"title":"Purification, crystallization and preliminary crystallographic analysis of Chlamydophila pneumoniae AP endonuclease IV","authors":"Yitong Zhang , Yangjie Ren , Ben Wang, Shiyang Guo, Siqi Wang, Jinglin Jin, Lihong Yang, Wei Gao","doi":"10.1016/j.pep.2024.106476","DOIUrl":"10.1016/j.pep.2024.106476","url":null,"abstract":"<div>Base excision is a crucial DNA repair process mediated by endonuclease IV in nucleotide excision. In Chlamydia pneumoniae, CpendoIV is the exclusive AP endonuclease IV, exhibiting DNA replication error-proofreading capabilities, making it a promising target for anti-chlamydial drug development. Predicting the structure of CpendoIV, molecular docking with DNA was performed, analyzing complex binding sites and protein surface electrostatic potential. Comparative structural studies were conducted with E. coli EndoIV and DNA complex containing AP sites.CpendoIV was cloned, expressed in E. coli, and purified via Ni-NTA chelation and size-exclusion chromatography. Low NaCl concentrations induced aggregation during purification, while high concentrations enhanced purity.CpendoIV recognizes and cleaving AP sites on dsDNA, and Zn2+ influences the activity. Crystallization was achieved under 8% (v/v) Tacsimate pH 5.2, 25% (w/v) polyethylene glycol 3350, and 1.91 Å resolution X-ray diffraction data was obtained at 100 K. This research is significant for provides a deeper understanding of CpendoIV involvement in the base excision repair process, offering insights into Chlamydia pneumoniae.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140194431","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

Affinity gel synthesis from the p-aminobenzoic acid derivative 4-amino-2-methylbenzoic acid and purification of polyphenol oxidase from various plant sources 对氨基苯甲酸衍生物 4-氨基-2-甲基苯甲酸的亲和凝胶合成及多种植物来源的多酚氧化酶的纯化。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-03-20 DOI: 10.1016/j.pep.2024.106474

Cansu Öztürk, Ömer İrfan Küfrevioğlu

{"title":"Affinity gel synthesis from the p-aminobenzoic acid derivative 4-amino-2-methylbenzoic acid and purification of polyphenol oxidase from various plant sources","authors":"Cansu Öztürk, Ömer İrfan Küfrevioğlu","doi":"10.1016/j.pep.2024.106474","DOIUrl":"10.1016/j.pep.2024.106474","url":null,"abstract":"<div>The polyphenol oxidase (PPO) enzyme, which causes enzymatic browning, has been repeatedly purified from fruit and vegetables by affinity chromatography. In the present research, Sepharose 4B-l-tyrosine-4-amino-2-methylbenzoic acid, a novel affinity gel for the purification of the PPO enzyme with high efficiency, was synthesized. Additionally, Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity gel, known in the literature, was also synthesized, and 9.02, 16.57, and 28.13 purification folds were obtained for the PPO enzymes of potato, mushroom, and eggplant by the reference gel. The PPO enzymes of potato, mushroom, and eggplant were purified 41.17, 64.47, and 56.78-fold from the new 4-amino-2-methylbenzoic acid gel. Following their isolation from the new affinity column, the assessment of PPO enzyme purity involved the utilization of SDS-PAGE. According to the results from SDS-PAGE and native PAGE, the molecular weight of each enzyme was 50 kDa. Then, the inhibition effects of naringin, morin hydrate, esculin hydrate, homovanillic acid, vanillic acid, phloridzin dihydrate, and p-coumaric acid phenolic compounds on purified potato, mushroom, and eggplant PPO enzyme were investigated. Among the tested phenolic compounds, morin hydrate was determined to be the most potent inhibitor on the potato (Ki: 0.07 ± 0.03 μM), mushroom (Ki: 0.7 ± 0.3 μM), and eggplant (Ki: 4.8 ± 1.2 μM) PPO enzymes. The studies found that the weakest inhibitor was homovanillic acid for the potato (Ki: 1112 ± 324 μM), mushroom (Ki: 567 ± 81 μM), and eggplant (Ki: 2016.7 ± 805.6 μM) PPO enzymes. Kinetic assays indicated that morin hydrate was a remarkable inhibitor on PPO.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140190094","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

Preparation and characterization of a novel humanized collagen III with repeated fragments of Gly300-Asp329 含有 Gly300-Asp329 重复片段的新型人源化胶原蛋白 III 的制备和表征。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-03-18 DOI: 10.1016/j.pep.2024.106473

Lingying Yan , Yao Zhang , Yuxiang Zhang , Qiexin Chen , Luyao Zhang , Xiao Han , Yumo Yang , Chun Zhang , Yongdong Liu , Rong Yu

{"title":"Preparation and characterization of a novel humanized collagen III with repeated fragments of Gly300-Asp329","authors":"Lingying Yan , Yao Zhang , Yuxiang Zhang , Qiexin Chen , Luyao Zhang , Xiao Han , Yumo Yang , Chun Zhang , Yongdong Liu , Rong Yu","doi":"10.1016/j.pep.2024.106473","DOIUrl":"10.1016/j.pep.2024.106473","url":null,"abstract":"<div>Recombinant human collagens have attracted intensive interest in the past two decades, demonstrating considerable potential in medicine, tissue engineering, and cosmetics. Several humanized recombinant collagens have been produced, exhibiting similar characteristics as the native species. To get insight into the structural and bioactive properties of different parts of collagen, in this study, the segment of Gly300-Asp329 of type III collagen was first adopted and repeated 18 times to prepare a novel recombinant collagen (named rhCLA). RhCLA was successfully expressed in E. coli, and a convenient separation procedure was established through reasonably combining alkaline precipitation and acid precipitation, yielding crude rhCLA with a purity exceeding 90%. Additionally, a polishing purification step utilizing cation exchange chromatography was developed, achieving rhCLA purity surpassing 98% and an overall recovery of approximately 120 mg/L culture. Simultaneously, the contents of endotoxin, nucleic acids, and host proteins were reduced to extremely low levels. This fragmented type III collagen displayed a triple-helical structure and gel-forming capability at low temperatures. Distinct fibrous morphology was also observed through TEM analysis. In cell experiments, rhCLA exhibited excellent biocompatibility and cell adhesion properties. These results provide valuable insights for functional studies of type III collagen and a reference approach for the large-scale production of recombinant collagens.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140176135","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

Escherichia coli as a versatile cell factory: Advances and challenges in recombinant protein production 作为多功能细胞工厂的大肠杆菌：重组蛋白质生产的进步与挑战。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-03-12 DOI: 10.1016/j.pep.2024.106463

İbrahim İncir , Özlem Kaplan

引用次数: 0

Genetically encoded site-specific 19F unnatural amino acid incorporation in V. natriegens for in-cell NMR analysis 将基因编码的特定位点 19F 非天然氨基酸掺入 V. natriegens，用于细胞内核磁共振分析。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-03-07 DOI: 10.1016/j.pep.2024.106461

Hao Li , Jin Zhang , Zilong Wang , Pan Shi , Chaowei Shi

引用次数: 0

Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras 以克鲁斯锥虫的查加斯蛋白为分子支架，将 TSA-1 的表位表达为可溶性重组嵌合体。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-02-27 DOI: 10.1016/j.pep.2024.106458

Rosa Elena Cárdenas-Guerra , Octavio Montes-Flores , Edgar Ezequiel Nava-Pintor , Gerardo Reséndiz-Cardiel , Claudia Ivonne Flores-Pucheta , Yasmín Irene Rodríguez-Gavaldón , Rossana Arroyo , Maria Elena Bottazzi , Peter J. Hotez , Jaime Ortega-López

{"title":"Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras","authors":"Rosa Elena Cárdenas-Guerra , Octavio Montes-Flores , Edgar Ezequiel Nava-Pintor , Gerardo Reséndiz-Cardiel , Claudia Ivonne Flores-Pucheta , Yasmín Irene Rodríguez-Gavaldón , Rossana Arroyo , Maria Elena Bottazzi , Peter J. Hotez , Jaime Ortega-López","doi":"10.1016/j.pep.2024.106458","DOIUrl":"10.1016/j.pep.2024.106458","url":null,"abstract":"<div>Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139997234","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