Protein expression and purification最新文献

{"title":"Purification, and characterization of detergent-compatible serine protease from Bacillus safensis strain PRN1: A sustainable alternative to hazardous chemicals in detergent industry","authors":"Panchi Rani Neog,&nbsp;Shubhangi Saini,&nbsp;Bolin Kumar Konwar","doi":"10.1016/j.pep.2024.106479","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106479","url":null,"abstract":"<div><p>Owing to vast therapeutic, commercial, and industrial applications of microbial proteases microorganisms from different sources are being explored. In this regard, the gut microbiota of <em>Monopterus</em> <em>cuchia</em> were isolated and examined for the production of protease. All the isolates were primarily and secondarily screened on skim milk and gelatin agar plates. The protease-positive isolates were characterized morphologically, biochemically, and molecularly. Out of the 20 isolated strains,6 belonging to five different genera viz<em>.</em> <em>Bacillus,</em> <em>Priestia,</em> <em>Aeromonas,</em> <em>Staphylococcus,</em> and <em>Serratia</em> demonstrated proteolytic activity. <em>Bacillus</em> <em>safensis</em> strain PRN1 demonstrated the highest protease production and, thus, the largest hydrolytic clear zones in both skim milk agar (15 ± 1 mm) and gelatin (16 ± 1 mm) plates. The optimized parameters (time, pH, temperature, carbon, nitrogen) for highest protease activity and microbial growth of <em>B.</em> <em>safensis</em> strain PRN1 includes 72 h (OD₆₀₀ = 0.56,1303 U/mL), pH 8 (OD₆₀₀ = 0.83, 403.29 U/mL), 40 °C (OD₆₀₀ = 1.75, 1849.11 U/mL), fructose (OD₆₀₀ = 1.22, 1502 U/mL), and gelatin (OD₆₀₀ = 1.88, 1015.33 U/mL). The enzyme was purified to homogeneity using salt-precipitation and gel filtration chromatography. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified enzyme was a monomer of a molecular weight of ∼33 kDa. The protease demonstrated optimal activity at pH 8 and 60 °C. It was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), demonstrating that it belongs to the serine-proteases family. The compatibility of the enzyme with surfactants and commercial detergents demonstrates its potential use in the detergent industry. Furthermore, the purified enzyme showed antibacterial and blood-stain removal properties.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"219 ","pages":"Article 106479"},"PeriodicalIF":1.6,"publicationDate":"2024-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140347855","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":"Characterization of a highly thermostable recombinant xylanase from Anoxybacillus ayderensis","authors":"Zuleyha Akpinar ,&nbsp;Hakan Karaoglu","doi":"10.1016/j.pep.2024.106478","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106478","url":null,"abstract":"<div><p>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 <em>Anoxybacillus ayderensis</em> as an intracellular xylanase <em>(AAy</em>XYN329) and recombinantly produced. While <em>AAy</em>XYN329 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. <em>Km</em>, <em>kcat</em> and <em>kcat</em>/<em>Km</em> values of <em>AAy</em>XYN329 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 <em>A. ayderensis</em> has an excellent potential to be utilized in many industrial processes.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"219 ","pages":"Article 106478"},"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}

{"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,&nbsp;Hosein Honari,&nbsp;Davoud Sadeghi,&nbsp;Hamid Bakhtiari","doi":"10.1016/j.pep.2024.106462","DOIUrl":"10.1016/j.pep.2024.106462","url":null,"abstract":"<div><p>The bacterium <em>Burkholderia pseudomallei</em> 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 <em>Escherichia coli</em> 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.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"219 ","pages":"Article 106462"},"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}

{"title":"High-yield and cost-effective biosynthesis process for producing antimicrobial peptide AA139","authors":"Ying Zhang ,&nbsp;Yapeng Wang ,&nbsp;Jianguang Lu ,&nbsp;Zongqing Huang ,&nbsp;Haoju Hua ,&nbsp;Yanan Li ,&nbsp;Jun Xu ,&nbsp;Jun Feng","doi":"10.1016/j.pep.2024.106475","DOIUrl":"10.1016/j.pep.2024.106475","url":null,"abstract":"<div><p>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 <em>Escherichia coli</em> (<em>E. coli</em>) 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.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"219 ","pages":"Article 106475"},"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}

{"title":"A two-step method preparation of semaglutide through solid-phase synthesis and inclusion body expression","authors":"Dezheng Peng ,&nbsp;Yang Li ,&nbsp;Linlin Si ,&nbsp;Bo Zhu ,&nbsp;Peng Wu ,&nbsp;Yibang Li ,&nbsp;Dongfang Tang ,&nbsp;Yu Liu ,&nbsp;Yunxiao Zhang","doi":"10.1016/j.pep.2024.106477","DOIUrl":"10.1016/j.pep.2024.106477","url":null,"abstract":"<div><p>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.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"219 ","pages":"Article 106477"},"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}

{"title":"Purification, crystallization and preliminary crystallographic analysis of Chlamydophila pneumoniae AP endonuclease IV","authors":"Yitong Zhang ,&nbsp;Yangjie Ren ,&nbsp;Ben Wang,&nbsp;Shiyang Guo,&nbsp;Siqi Wang,&nbsp;Jinglin Jin,&nbsp;Lihong Yang,&nbsp;Wei Gao","doi":"10.1016/j.pep.2024.106476","DOIUrl":"10.1016/j.pep.2024.106476","url":null,"abstract":"<div><p>Base excision is a crucial DNA repair process mediated by endonuclease IV in nucleotide excision. In <em>Chlamydia pneumoniae</em>, <em>Cp</em>endoIV 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 <em>Cp</em>endoIV, molecular docking with DNA was performed, analyzing complex binding sites and protein surface electrostatic potential. Comparative structural studies were conducted with <em>E. coli</em> EndoIV and DNA complex containing AP sites.<em>Cp</em>endoIV was cloned, expressed in <em>E. coli</em>, and purified via Ni-NTA chelation and size-exclusion chromatography. Low NaCl concentrations induced aggregation during purification, while high concentrations enhanced purity.<em>Cp</em>endoIV 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 <em>Cp</em>endoIV involvement in the base excision repair process, offering insights into <em>Chlamydia pneumoniae</em>.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"219 ","pages":"Article 106476"},"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}

{"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,&nbsp;Ömer İrfan Küfrevioğlu","doi":"10.1016/j.pep.2024.106474","DOIUrl":"10.1016/j.pep.2024.106474","url":null,"abstract":"<div><p>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-<span>l</span>-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-<span>l</span>-tyrosine-<em>p-</em>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 (K_i: 0.07 ± 0.03 μM), mushroom (K_i: 0.7 ± 0.3 μM), and eggplant (K_i: 4.8 ± 1.2 μM) PPO enzymes. The studies found that the weakest inhibitor was homovanillic acid for the potato (K_i: 1112 ± 324 μM), mushroom (K_i: 567 ± 81 μM), and eggplant (K_i: 2016.7 ± 805.6 μM) PPO enzymes. Kinetic assays indicated that morin hydrate was a remarkable inhibitor on PPO.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"219 ","pages":"Article 106474"},"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}

{"title":"Preparation and characterization of a novel humanized collagen III with repeated fragments of Gly300-Asp329","authors":"Lingying Yan ,&nbsp;Yao Zhang ,&nbsp;Yuxiang Zhang ,&nbsp;Qiexin Chen ,&nbsp;Luyao Zhang ,&nbsp;Xiao Han ,&nbsp;Yumo Yang ,&nbsp;Chun Zhang ,&nbsp;Yongdong Liu ,&nbsp;Rong Yu","doi":"10.1016/j.pep.2024.106473","DOIUrl":"10.1016/j.pep.2024.106473","url":null,"abstract":"<div><p>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 <em>E. coli</em>, 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.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"219 ","pages":"Article 106473"},"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}

{"title":"Escherichia coli as a versatile cell factory: Advances and challenges in recombinant protein production","authors":"İbrahim İncir ,&nbsp;Özlem Kaplan","doi":"10.1016/j.pep.2024.106463","DOIUrl":"10.1016/j.pep.2024.106463","url":null,"abstract":"<div><p><em>E. coli</em> plays a substantial role in recombinant protein production. Its importance increased with the discovery of recombinant DNA technology and the subsequent production of the first recombinant insulin in <em>E. coli</em>. <em>E. coli</em> is a widely used and cost-effective host to produce recombinant proteins. It is also noteworthy that a significant portion of the approved therapeutic proteins have been produced in this organism<em>.</em> Despite these advantages, it has some disadvantages, such as toxicity and lack of eukaryotic post-translational modifications that can lead to the production of misfolded, insoluble, or dysfunctional proteins.</p><p>This study focused on the challenges and engineering approaches for improved expression and solubility in recombinant protein production in <em>E. coli</em>. In this context, solution strategies such as strain and vector selection, codon usage, mRNA stability, expression conditions, translocation to the periplasmic region and addition of fusion tags in <em>E. coli</em> were discussed.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"219 ","pages":"Article 106463"},"PeriodicalIF":1.6,"publicationDate":"2024-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140120439","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":"Genetically encoded site-specific 19F unnatural amino acid incorporation in V. natriegens for in-cell NMR analysis","authors":"Hao Li ,&nbsp;Jin Zhang ,&nbsp;Zilong Wang ,&nbsp;Pan Shi ,&nbsp;Chaowei Shi","doi":"10.1016/j.pep.2024.106461","DOIUrl":"10.1016/j.pep.2024.106461","url":null,"abstract":"<div><p>Nuclear magnetic resonance (NMR) spectroscopy NMR is a well-established technique for probing protein structure, dynamics and conformational changes. Taking advantage of the high signal sensitivity and broad chemical shift range of ¹⁹F nuclei, ¹⁹F NMR has been applied to investigate protein function at atomic resolution. In this report, we extend the unnatural amino acid site-specific incorporation into <em>V. natriegens</em>, an alternate protein expression system. The unnatural amino acid L-4-trifluoromethylphenylalanine (tfmF) was site-specifically introduced into the mitogen-activated protein kinase MEKK3 in <em>V. natriegens</em> using genetically encoded technology<em>,</em> which will be an extensive method for in-cell protein structure and dynamic investigation.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"219 ","pages":"Article 106461"},"PeriodicalIF":1.6,"publicationDate":"2024-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140068657","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}