Protein Engineering, Design and Selection最新文献_第3页

Modification of the peroxygenative: peroxidative activity ratio in the unspecific peroxygenase from Agrocybe aegerita by structure-guided evolution 结构导向进化对绿草草非特异性过氧酶过氧活性比的修饰

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzw073

D. Maté, M. A. Palomino, Patricia Molina-Espeja, Javier Martin-Diaz, M. Alcalde

{"title":"Modification of the peroxygenative: peroxidative activity ratio in the unspecific peroxygenase from Agrocybe aegerita by structure-guided evolution","authors":"D. Maté, M. A. Palomino, Patricia Molina-Espeja, Javier Martin-Diaz, M. Alcalde","doi":"10.1093/protein/gzw073","DOIUrl":"https://doi.org/10.1093/protein/gzw073","url":null,"abstract":"Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase capable of performing with high-selectivity C-H oxyfunctionalizations of great interest in organic synthesis through its peroxygenative activity. However, the convergence of such activity with an unwanted peroxidative activity encumbers practical applications. In this study, we have modified the peroxygenative:peroxidative activity ratio (P:p ratio) of UPO from Agrocybe aegerita by structure-guided evolution. Several flexible loops (Glu1-Pro35, Gly103-Asp131, Ser226-Gly243, Gln254-Thr276 and Ty293-Arg327) were selected on the basis on their B-factors and ΔΔG values. The full ensemble of segments (43% of UPO sequence) was subjected to focused evolution by the Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) method in Saccharomyces cerevisiae. Five independent mutant libraries were screened in terms of P:p ratio and thermostability. We identified several variants that harbored substitutions at positions 120 and 320 with a strong enhancement in the P:p ratio albeit at the cost of stability. The most thermostable mutant of this process (S226G with an increased T50 of 2°C) was subjected to further combinatorial saturation mutagenesis on Thr120 and Thr320 yielding a collection of variants with modified P:p ratio and recovered stability. Our results seem to indicate the coexistence of several oxidation sites for peroxidative and peroxygenative activities in UPO.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81774134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 24

Probing the influence of non-covalent contact networks identified by charge density analysis on the oxidoreductase BacC 探讨电荷密度分析鉴定的非共价接触网络对氧化还原酶BacC的影响

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzx006

K. Perinbam, H. Balaram, T. N. Guru Row, B. Gopal

{"title":"Probing the influence of non-covalent contact networks identified by charge density analysis on the oxidoreductase BacC","authors":"K. Perinbam, H. Balaram, T. N. Guru Row, B. Gopal","doi":"10.1093/protein/gzx006","DOIUrl":"https://doi.org/10.1093/protein/gzx006","url":null,"abstract":"Bacillus subtilis BacC is an oxidoreductase involved in the biosynthesis of the potent antibiotic bacilysin. The crystal structure of BacC was determined at 1.19 Å resolution. An experimental charge density approach was used to calculate non-covalent interactions within the monomer and across the dimeric interface of BacC. This interaction network, in turn, enabled an analysis of non-covalently connected paths that span the protein structure. One of the pathways of non-covalent interactions was examined by mutational analysis. Biochemical analysis of BacC mutants with potential disruptions in non-covalent interactions along this path revealed that residues that form nodes in pathways of non-covalent interactions influence catalytic activity more than others in a similar chemical environment. Furthermore, we note that nodes in the non-covalent interaction networks are co-localized with compensatory mutation sites identified by multiple sequence alignment of proteins with low sequence similarity to BacC. Put together, this analysis supports the hypothesis that non-covalent nodes represent conserved structural features that can impact the catalytic activity of an enzyme.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78795870","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 4

Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1 墨西哥利什曼原虫scp2 -硫酶(2型)的晶体学底物结合研究:氧阴离子孔-1的独特特征

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzw080

R. Harijan, T. Kiema, Shahan M Syed, Imran Qadir, M. Mazet, F. Bringaud, P. Michels, R. Wierenga

{"title":"Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1","authors":"R. Harijan, T. Kiema, Shahan M Syed, Imran Qadir, M. Mazet, F. Bringaud, P. Michels, R. Wierenga","doi":"10.1093/protein/gzw080","DOIUrl":"https://doi.org/10.1093/protein/gzw080","url":null,"abstract":"C\u0000Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-thiolase (type-2) (Lm-thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-thiolase. The amino acid sequence fingerprints ( xS, EAF, G P) of three catalytic loops identify the active site geometry of the well-studied CNH-thiolases, whereas SCP2-thiolases (type-1, type-2) are classified as CHH-thiolases, having as corresponding fingerprints xS, DCF and G P. In all thiolases, OAH2 is formed by the main chain NH groups of two catalytic loops. In the well-studied CNH-thiolases, OAH1 is formed by a water (of the Wat-Asn(NEAF) dyad) and NE2 (of the GHP-histidine). In the two described liganded Lm-thiolase structures, it is seen that in this CHH-thiolase, OAH1 is formed by NE2 of His338 (HDCF) and His388 (GHP). Analysis of the OAH1 hydrogen bond networks suggests that the GHP-histidine is doubly protonated and positively charged in these complexes, whereas the HDCF histidine is neutral and singly protonated.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84007099","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Collective repacking reveals that the structures of protein cores are uniquely specified by steric repulsive interactions 集体重新包装揭示了蛋白质核心的结构是由空间排斥性相互作用唯一指定的

Protein Engineering, Design and Selection Pub Date : 2017-02-15 DOI: 10.1093/protein/gzx011

Jennifer C. Gaines, Alejandro Virrueta, D. A. Buch, S. Fleishman, C. O’Hern, Lynne Regan

{"title":"Collective repacking reveals that the structures of protein cores are uniquely specified by steric repulsive interactions","authors":"Jennifer C. Gaines, Alejandro Virrueta, D. A. Buch, S. Fleishman, C. O’Hern, Lynne Regan","doi":"10.1093/protein/gzx011","DOIUrl":"https://doi.org/10.1093/protein/gzx011","url":null,"abstract":"Abstract Protein core repacking is a standard test of protein modeling software. A recent study of six different modeling software packages showed that they are more successful at predicting side chain conformations of core compared to surface residues. All the modeling software tested have multicomponent energy functions, typically including contributions from solvation, electrostatics, hydrogen bonding and Lennard–Jones interactions in addition to statistical terms based on observed protein structures. We investigated to what extent a simplified energy function that includes only stereochemical constraints and repulsive hard-sphere interactions can correctly repack protein cores. For single residue and collective repacking, the hard-sphere model accurately recapitulates the observed side chain conformations for Ile, Leu, Phe, Thr, Trp, Tyr and Val. This result shows that there are no alternative, sterically allowed side chain conformations of core residues. Analysis of the same set of protein cores using the Rosetta software suite revealed that the hard-sphere model and Rosetta perform equally well on Ile, Leu, Phe, Thr and Val; the hard-sphere model performs better on Trp and Tyr and Rosetta performs better on Ser. We conclude that the high prediction accuracy in protein cores obtained by protein modeling software and our simplified hard-sphere approach reflects the high density of protein cores and dominance of steric repulsion.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73163427","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

Engineering potent long-acting variants of the Wnt inhibitor DKK2 设计有效的长效Wnt抑制剂DKK2变体

Protein Engineering, Design and Selection Pub Date : 2017-02-09 DOI: 10.1093/protein/gzx007

Richelle Sopko, Joshua W. Mugford, A. Lehmann, R. Shapiro, M. Rushe, Abhishek Kulkarni, Joe Worrall, Joseph Amatucci, Dingyi Wen, N. Pederson, Brenda K. Minesinger, J. Arndt, B. Pepinsky

{"title":"Engineering potent long-acting variants of the Wnt inhibitor DKK2","authors":"Richelle Sopko, Joshua W. Mugford, A. Lehmann, R. Shapiro, M. Rushe, Abhishek Kulkarni, Joe Worrall, Joseph Amatucci, Dingyi Wen, N. Pederson, Brenda K. Minesinger, J. Arndt, B. Pepinsky","doi":"10.1093/protein/gzx007","DOIUrl":"https://doi.org/10.1093/protein/gzx007","url":null,"abstract":"Abstract Wnt signaling pathways are required for a wide variety of biological processes ranging from embryonic development to tissue repair and regeneration. Dickkopf-2 (DKK2) is classically defined as a canonical Wnt inhibitor, though it may play a role in activating non-canonical Wnt pathways in the context of endothelial network formation after acute injury. Here we report the discovery of a fusion partner for a DKK2 polypeptide that significantly improves the expression, biochemical properties and pharmacokinetics (PK) of the DKK2 polypeptide. Specifically, human serum albumin (HSA) was identified as a highly effective fusion partner. Substitution of selected amino acid residues in DKK2 designed to decrease heparan sulfate binding by HSA-DKK2 variants, further improved the PK properties of the molecule in rodents. The HSA-DKK2 variants were monomeric, as thermally stable as wild type, and active as measured by their ability to bind to and prevent phosphorylation of the Wnt coreceptor LRP6. Our engineering efforts resulted in potent long-lived variants of the canonical Wnt inhibitor DKK2, applicable for Wnt pathway manipulation either by systematic delivery or focused administration at sites of tissue injury.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76004797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers 克服高效神经毒剂生物清除剂定向进化中的优化平台期

Protein Engineering, Design and Selection Pub Date : 2017-02-04 DOI: 10.1093/protein/gzx003

M. Goldsmith, N. Aggarwal, Y. Ashani, H. Jubran, Per Greisen, S. Ovchinnikov, H. Leader, D. Baker, J. Sussman, A. Goldenzweig, S. Fleishman, Dan S. Tawfik

{"title":"Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers","authors":"M. Goldsmith, N. Aggarwal, Y. Ashani, H. Jubran, Per Greisen, S. Ovchinnikov, H. Leader, D. Baker, J. Sussman, A. Goldenzweig, S. Fleishman, Dan S. Tawfik","doi":"10.1093/protein/gzx003","DOIUrl":"https://doi.org/10.1093/protein/gzx003","url":null,"abstract":"Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau. We aimed to optimize the catalytic efficiency of bacterial phosphotriesterase (PTE) toward V-type nerve agents. Previously, we improved the catalytic efficiency of wild-type PTE toward the nerve agent VX by 500-fold, to a catalytic efficiency (kcat/KM) of 5 × 106 M-1 min-1. However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of >107 M-1 min-1. Here, following eight additional rounds of directed evolution and the computational design of a stabilized variant, we evolved PTE variants that detoxify VX with a kcat/KM ≥ 5 × 107 M-1 min-1 and Russian VX (RVX) with a kcat/KM ≥ 107 M-1 min-1. These final 10-fold improvements were the most time consuming and laborious, as most libraries yielded either minor or no improvements. Stabilizing the evolving enzyme, and avoiding tradeoffs in activity with different substrates, enabled us to obtain further improvements beyond the optimization plateau and evolve PTE variants that were overall improved by >5000-fold with VX and by >17 000-fold with RVX. The resulting variants also hydrolyze G-type nerve agents with high efficiency (GA, GB at kcat/KM > 5 × 107 M-1 min-1) and can thus serve as candidates for broad-spectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85375983","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 49

Annexin-directed &bgr;-glucuronidase for the targeted treatment of solid tumors 膜联蛋白导向的葡糖苷酶用于实体瘤的靶向治疗

Protein Engineering, Design and Selection Pub Date : 2017-02-01 DOI: 10.1093/protein/gzw063

Katrin P. Guillen, E. Ruben, N. Virani, R. Harrison

{"title":"Annexin-directed &bgr;-glucuronidase for the targeted treatment of solid tumors","authors":"Katrin P. Guillen, E. Ruben, N. Virani, R. Harrison","doi":"10.1093/protein/gzw063","DOIUrl":"https://doi.org/10.1093/protein/gzw063","url":null,"abstract":"Enzyme prodrug therapy has the potential to remedy the lack of selectivity associated with the systemic administration of chemotherapy. However, most current systems are immunogenic and constrained to a monotherapeutic approach. We developed a new class of fusion proteins centered about the human enzyme &bgr;-glucuronidase (&bgr;G), capable of converting several innocuous prodrugs into chemotherapeutics. We targeted &bgr;G to phosphatidylserine on tumor cells, tumor vasculature and metastases via annexin A1/A5. Phosphatidylserine shows promise as a universal marker for solid tumors and allows for tumor type-independent targeting. To create fusion proteins, human annexin A1/A5 was genetically fused to the activity-enhancing 16a3 mutant of human &bgr;G, expressed in chemically defined, fed-batch suspension culture, and chromatographically purified. All fusion constructs achieved >95% purity with yields up to 740 &mgr;g/l. Fusion proteins displayed cancer selective cell-surface binding with cell line-dependent binding stability. One fusion protein in combination with the prodrug SN-38 glucuronide was as effective as the drug SN-38 on Panc-1 pancreatic cancer cells and HAAE-1 endothelial cells, and demonstrated efficacy against MCF-7 breast cancer cells. &bgr;G fusion proteins effectively enable localized combination therapy that can be tailored to each patient via prodrug selection, with promising clinical potential based on their near fully human design.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85052688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Directed evolution of P450cin for mediated electron transfer P450cin介导电子转移的定向进化

Protein Engineering, Design and Selection Pub Date : 2017-02-01 DOI: 10.1093/protein/gzw072

Ketaki D. Belsare, Thomas Horn, A. J. Ruff, Ronny Martínez, Anders O. Magnusson, D. Holtmann, J. Schrader, U. Schwaneberg

{"title":"Directed evolution of P450cin for mediated electron transfer","authors":"Ketaki D. Belsare, Thomas Horn, A. J. Ruff, Ronny Martínez, Anders O. Magnusson, D. Holtmann, J. Schrader, U. Schwaneberg","doi":"10.1093/protein/gzw072","DOIUrl":"https://doi.org/10.1093/protein/gzw072","url":null,"abstract":"Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C–H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions. However, natural enzymes are often not suitable for using non-natural electron delivery systems. Here we report the directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III)sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of Sequence Saturation Mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generated a variant (Gln385His, Val386Ser, Thr77Asn, Leu88Arg; named KB8) with a 3.8-fold increase in catalytic efficiency (28 µM−1 min−1) compared to P450cin-ADD-CinC (7 µM−1 min−1). Furthermore, variant KB8 exhibited a 1.5-fold higher product formation (500 µM µM−1 P450) compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as co-factor (315 µM µM−1 P450). In addition, electrochemical experiments with the electron delivery system platinum/cobalt(III)sepulchrate showed that the KB8 variant had a 4-fold higher product formation rate (0.16 nmol (nmol) P450−1 min−1 cm−2) than the P450cin-ADD-CinC (0.04 nmol (nmol) P450−1 min−1 cm−2). In summary, the current work shows prospects of using directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87335022","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 16

Deep sequencing of phage-displayed peptide libraries reveals sequence motif that detects norovirus. 噬菌体展示肽库的深度测序揭示了检测诺如病毒的序列基序。

Protein Engineering, Design and Selection Pub Date : 2017-02-01 Epub Date: 2016-12-28 DOI: 10.1093/protein/gzw074

Amy M Hurwitz, Wanzhi Huang, Mary K Estes, Robert L Atmar, Timothy Palzkill

{"title":"Deep sequencing of phage-displayed peptide libraries reveals sequence motif that detects norovirus.","authors":"Amy M Hurwitz, Wanzhi Huang, Mary K Estes, Robert L Atmar, Timothy Palzkill","doi":"10.1093/protein/gzw074","DOIUrl":"10.1093/protein/gzw074","url":null,"abstract":"<p><p>Norovirus infections are the leading cause of non-bacterial gastroenteritis and result in about 21 million new cases and $2 billion in costs per year in the United States. Existing diagnostics have limited feasibility for point-of-care applications, so there is a clear need for more reliable, rapid, and simple-to-use diagnostic tools in order to contain outbreaks and prevent inappropriate treatments. In this study, a combination of phage display technology, deep sequencing and computational analysis was used to identify 12-mer peptides with specific binding to norovirus genotype GI.1 virus-like particles (VLPs). After biopanning, phage populations were sequenced and analyzed to identify a consensus peptide motif-YRSWXP. Two 12-mer peptides containing this sequence, NV-O-R5-3 and NV-O-R5-6, were further characterized to evaluate the motif's functional ability to detect VLPs and virus. Results indicated that these peptides effectively detect GI.1 VLPs in solid-phase peptide arrays, ELISAs and dot blots. Further, their specificity for the S-domain of the major capsid protein enables them to detect a wide range of GI and GII norovirus genotypes. Both peptides were able to detect virus in norovirus-positive clinical stool samples. Overall, the work reported here demonstrates the application of phage display coupled with next generation sequencing and computational analysis to uncover peptides with specific binding ability to a target protein for diagnostic applications. Further, the reagents characterized here can be integrated into existing diagnostic formats to detect clinically relevant genotypes of norovirus in stool.</p>","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzw074","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83155832","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 8

Production and characterization of functional recombinant hybrid heteropolymers of camel hepcidin and human ferritin H and L chains 骆驼铁蛋白与人铁蛋白H链、L链功能性杂交物的制备与表征

Protein Engineering, Design and Selection Pub Date : 2017-02-01 DOI: 10.1093/protein/gzw066

Mohamed Boumaiza, Fernando Carmona, M. Poli, M. Asperti, A. Gianoncelli, Michela Bertuzzi, Paola Ruzzenenti, P. Arosio, M. Marzouki

{"title":"Production and characterization of functional recombinant hybrid heteropolymers of camel hepcidin and human ferritin H and L chains","authors":"Mohamed Boumaiza, Fernando Carmona, M. Poli, M. Asperti, A. Gianoncelli, Michela Bertuzzi, Paola Ruzzenenti, P. Arosio, M. Marzouki","doi":"10.1093/protein/gzw066","DOIUrl":"https://doi.org/10.1093/protein/gzw066","url":null,"abstract":"Hepcidin is a liver-synthesized hormone that plays a central role in the regulation of systemic iron homeostasis. To produce a new tool for its functional properties the cDNA coding for camel hepcidin-25 was cloned at the 5’end of human FTH sequence into the pASK-IBA43plus vector for expression in Escherichia coli. The recombinant fusion hepcidin–ferritin-H subunit was isolated as an insoluble iron-containing protein. When alone it did not refold in a 24-mer ferritin molecule, but it did when renatured together with H- or L-ferritin chains. We obtained stable ferritin shells exposing about 4 hepcidin peptides per 24-mer shell. The molecules were then reduced and re-oxidized in a controlled manner to allow the formation of the proper hepcidin disulfide bridges. The functionality of the exposed hepcidin was confirmed by its ability to specifically bind the mouse macrophage cell line J774 that express ferroportin and to promote ferroportin degradation. This chimeric protein may be useful for studying the hepcidin–ferroportin interaction in cells and also as drug-delivery agent.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79942638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7