Protein expression and purification最新文献

{"title":"Unexpected tobacco etch virus (TEV) protease cleavage of recombinant human proteins","authors":"Lauren P. Beaumont,&nbsp;Jennifer Mehalko,&nbsp;Adam Johnson,&nbsp;Vanessa E. Wall,&nbsp;Dominic Esposito","doi":"10.1016/j.pep.2024.106488","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106488","url":null,"abstract":"<div><p>The tobacco etch virus (TEV) protease is a commonly used reagent for removal of solubility and purification tags from recombinant proteins and is cited as being highly specific for its canonical cleavage site. Flexibility in some amino acids within this recognition sequence has been described in the literature but researchers generally assume few native human proteins will carry off-target sequences for TEV cleavage. We report here the aberrant cleavage of three human proteins with non-canonical TEV protease cleavage sites and identify broader sequence specificity rules that can be used to predict unwanted cleavage of recombinant proteins. Using these rules, 456 human proteins were identified that could be substrates for unwanted TEV protease cleavage.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140818333","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 preliminary crystallographic analysis of bacterial transmembrane protein EfeU for iron import","authors":"Kenji Okumura ,&nbsp;Bunzo Mikami ,&nbsp;Sayoko Oiki ,&nbsp;Kohei Ogura ,&nbsp;Wataru Hashimoto","doi":"10.1016/j.pep.2024.106487","DOIUrl":"10.1016/j.pep.2024.106487","url":null,"abstract":"<div><p>The bacterial Efe system functions as an importer of free Fe²⁺ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from <em>Escherichia coli</em>, selected <em>E. coli</em> Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140788515","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 and biochemical characterization of a novel thermostable alkaline β-1,3–1,4-glucanase (lichenase) from an alkaliphilic Bacillus lehensis G1","authors":"Noor Liana Mat Yajit ,&nbsp;Noor Haza Fazlin Hashim ,&nbsp;Rosli Mohd Illias ,&nbsp;Abdul Munir Abdul Murad","doi":"10.1016/j.pep.2024.106486","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106486","url":null,"abstract":"<div><p>New thermostable β-1,3–1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria <em>Bacillus lehensis</em> G1. The genome sequence of <em>B. lehensis</em> predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for <em>Blg29</em> was successfully amplified via PCR and subsequently expressed as a recombinant protein using the <em>E. coli</em> expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca²⁺. Blg29 showed high substrate activity towards lichenan where <em>V</em>_m, <em>K</em>_m, <em>K</em>_cat, and <em>k</em>_cat/<em>K</em>_m values were 2040.82 μmolmin^‾1mg^‾1, 4.69 mg/mL, and 986.39 s‾¹ and 210.32 mLs^‾1mg‾¹ respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140633196","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":"Design and construction of a phage-displayed Camelid nanobody library using a simple bioinformatics method","authors":"Aliasghar Rahimian ,&nbsp;Ali Nabati ,&nbsp;Hooman Askari ,&nbsp;Mohammad Saffarioun ,&nbsp;Mahdi Aminian","doi":"10.1016/j.pep.2024.106485","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106485","url":null,"abstract":"<div><h3>Background</h3><p>Rational design of synthetic phage-displayed libraries requires the identification of the most appropriate positions for randomization using defined amino acid sets to recapitulate the natural occurrence. The present study uses position-specific scoring matrixes (PSSMs) for identifying and randomizing Camelidae nanobody (VHH) CDR3. The functionality of a synthetic VHH repertoire designed by this method was tested for discovering new VHH binders to recombinant coagulation factor VII (rfVII).</p></div><div><h3>Methods</h3><p>Based on PSSM analysis, the CDR3 of cAbBCII10 VHH framework was identified, and a set of amino acids for the substitution of each PSSM-CDR3 position was defined. Using the Rosetta design SwiftLib tool, the final repertoire was back-translated to a degenerate nucleotide sequence. A synthetic phage-displayed library was constructed based on this repertoire and screened for anti-rfVII binders.</p></div><div><h3>Results</h3><p>A synthetic phage-displayed VHH library with 1 × 10⁸ variants was constructed. Three VHH binders to rfVII were isolated from this library with estimated dissociation constants (K_D) of 1 × 10⁻⁸ M, 5.8 × 10⁻⁸ M and 2.6 × 10^-⁷ M.</p></div><div><h3>Conclusion</h3><p>PSSM analysis is a simple and efficient way to design synthetic phage-displayed libraries.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140647084","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":"Alocasia macrorrhiza rhizome lectin inhibits growth of pathogenic bacteria and human lung cancer cell in vitro and Ehrlich ascites carcinoma cell in vivo in mice","authors":"Hossain Mohammad Hridoy ,&nbsp;Md Pervez Hossain ,&nbsp;Md Hasan Ali ,&nbsp;Imtiaj Hasan ,&nbsp;Md Belal Uddin ,&nbsp;Mohammad Taufiq Alam ,&nbsp;Syed Rashel Kabir","doi":"10.1016/j.pep.2024.106484","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106484","url":null,"abstract":"<div><p>Cancer and antibiotic resistance represent significant global challenges, affecting public health and healthcare systems worldwide. Lectin, a carbohydrate-binding protein, displays various biological properties, including antimicrobial and anticancer activities. This study focused on anticancer and antibacterial properties of <em>Alocasia macrorrhiza</em> lectin (AML). AML, with a molecular weight of 11.0 ± 1.0 kDa was purified using Ion-exchange chromatography, and the homotetrameric form was detected by gel-filtration chromatography. It agglutinates mouse erythrocytes, that was inhibited by 4-Nitrophenyl-α-<span>d</span>-mannopyranoside. Maximum hemagglutination activity was observed below 60 °C and within a pH range from 8 to 11. Additionally, it exhibited moderate toxicity against brine shrimp nauplii with LD₅₀ values of 321 μg/ml and showed antibacterial activity against <em>Escherichia coli</em> and <em>Shigella dysenteriae</em>. <em>In vitro</em> experiments demonstrated that AML suppressed the proliferation of mice Ehrlich ascites carcinoma (EAC) cells by 35 % and human lung cancer (A549) cells by 40 % at 512 μg/ml concentration. <em>In vivo</em> experiments involved intraperitoneal injection of AML in EAC-bearing mice for five consecutive days at doses of 2.5 and 5.0 mg/kg/day, and the results indicated that AML inhibited EAC cell growth by 37 % and 54 %, respectively. Finally, it can be concluded that AML can be used for further anticancer and antibacterial studies.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140558240","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":"Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins – A case study for Mefp-3 from Mytilus edulis","authors":"Constanze Zwies ,&nbsp;Ángela María Vargas Rodríguez ,&nbsp;Marcel Naumann ,&nbsp;Franziska Seifert ,&nbsp;Markus Pietzsch","doi":"10.1016/j.pep.2024.106483","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106483","url":null,"abstract":"<div><p>Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). <em>Mytilus edulis</em> foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from <em>Verrucomicrobium spinosum</em> was used for the <em>in vitro</em> hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S104659282400055X/pdfft?md5=d9688fe00176c5d4d32973db47e6eafc&pid=1-s2.0-S104659282400055X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140555386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expression, purification, and crystal structure of mpox virus A41 protein","authors":"Haihai Jiang ,&nbsp;Juncheng Li ,&nbsp;Yuxin Jian ,&nbsp;Tingting Yang ,&nbsp;Jin Zhang ,&nbsp;Jian Li","doi":"10.1016/j.pep.2024.106480","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106480","url":null,"abstract":"<div><p>Mpox is a zoonotic disease that was once endemic in Africa countries caused by mpox virus. However, cases recently have been confirmed in many non-endemic countries outside of Africa. The rapidly increasing number of confirmed mpox cases poses a threat to the international community. In-depth studies of key viral factors are urgently needed, which will inform the design of multiple antiviral agents. Mpox virus <em>A41L</em> gene encodes a secreted protein, A41, that is nonessential for viral replication, but could affect the host response to infection via interacting with chemokines. Here, mpox virus A41 protein was expressed in Sf9 cells, and purified by affinity chromatography followed by gel filtration. Surface plasmon resonance spectroscopy showed that purified A41 binds a certain human chemokine CXCL8 with the equilibrium dissociation constant (<em>K</em>_D) being 1.22 × 10⁻⁶ M. The crystal structure of mpox virus A41 protein was solved at 1.92 Å. Structural analysis and comparison revealed that mpox virus A41 protein adopts a characteristic β-sheet topology, showing minor differences with that of vaccinia virus. These preliminary structural and functional studies of A41 protein from mpox virus will help us better understand its role in chemokine subversion, and contributing to the knowledge to viral chemokine binding proteins.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140558241","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 calcium-binding photoprotein clytin II: Expression of the preferred human codon-optimized clytin II gene in Chinese hamster ovary-K1 cells and its use in the G-protein-coupled receptor assays","authors":"Satoshi Inouye ,&nbsp;Jun-ichi Sato ,&nbsp;Yuiko Sahara-Miura ,&nbsp;Sunao Hisada","doi":"10.1016/j.pep.2024.106481","DOIUrl":"10.1016/j.pep.2024.106481","url":null,"abstract":"<div><p>Clytin II (CLII) is a Ca²⁺-binding photoprotein and has been identified as an isotype of clytin I (CLI). CLII consists of apoCLII (an apoprotein) and 2-peroxide of coelenterazine (an adduct of molecular oxygen to coelenterazine), which is identical to the widely used Ca²⁺-binding photoprotein, aequorin (AQ). However, CLII triggered by Ca²⁺ exhibits a 4.5-fold higher maximum luminescence intensity (<em>I</em>_max) compared to both AQ and CLI, and it is approximately 5 times less sensitive to Ca²⁺ than AQ. To confirm the suitability of the preferred human codon-optimized CLII (pCLII) gene for cell-based G-protein-coupled receptor (GPCR) assays, a transformant stably expressing apoprotein of pCLII using the pCLII gene in the mitochondria of CHO–K1 cells was established and <em>in situ</em> regenerated pCLII in the cells were applied to the high-throughput screening system. An ATP-stimulated GPCR assay for endogenous P2Y purinergic receptors was confirmed using the established stable transformant.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140760159","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":"Enzymatic characterization and thermostability improvement of an acidophilic endoxylanase PphXyn11 from Paenibacillus physcomitrellae XB","authors":"Le Wang,&nbsp;Yan Yan Wang,&nbsp;Zhi Ling Chen,&nbsp;Yan Hong Li","doi":"10.1016/j.pep.2024.106482","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106482","url":null,"abstract":"<div><p>GH11 enzyme is known to be specific and efficient for the hydrolysis of xylan. It has been isolated from many microorganisms, and its enzymatic characteristics and thermostability vary between species. In this study, a GH11 enzyme PphXyn11 from a novel xylan-degrading strain of <em>Paenibacillus physcomitrellae</em> XB was characterized, and five mutants were constructed to try to improve the enzyme's thermostability. The results showed that PphXyn11 was an acidophilic <em>endo</em>-<em>β</em>-1,4-xylanase with the optimal reaction pH of 3.0–4.0, and it could deconstruct different kinds of xylan substrates efficiently, such as beechwood xylan, wheat arabinoxylan and xylo-oligosaccharides, to produce xylobiose and xylotriose as the main products at the optimal reaction temperature of 40 °C. Improvement of the thermal stability of PphXyn11 using site-directed mutagenesis revealed that three mutants, W33C/N47C, S127C/N174C and S49E, designed by adding the disulfide bonds at the <em>N</em>-terminal, <em>C</em>-terminal and increasing the charged residues on the surface of PphXyn11 respectively, could increase the enzymatic activity and thermal stablility significantly and make the optimal reaction temperature reach 50 °C. Molecular dynamics simulations as well as computed the numbers of salt bridges and hydrogen bonds indicated that the protein structures of these three mutants were more stable than the wild type, which provided theoretical support for their improved thermal stability. Certainly, further research is necessary to improve the enzymatic characteristics of PphXyn11 to achieve the bioconversion of hemicellulosic biomass on an applicable scale.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140535023","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, 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":null,"pages":null},"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}