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Pesquisa : G02.111.570.060.440 [Categoria DeCS]
Referências encontradas : 539 [refinar]
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  1 / 539 MEDLINE  
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[PMID]:28649707
[Au] Autor:Ramsoomair CK; Yakely AE; Urbanski LM; Karanja K; Giaccone ZT; Siegart NM; Wang C; Gomez AV; Reitter JN; Mills KV
[Ad] Endereço:Department of Chemistry, College of the Holy Cross, Worcester, MA, USA.
[Ti] Título:Coordination of the third step of protein splicing in two cyanobacterial inteins.
[So] Source:FEBS Lett;591(14):2147-2154, 2017 Jul.
[Is] ISSN:1873-3468
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The third step of protein splicing is cyclization of Asn coupled to peptide bond cleavage. In two related cyanobacterial inteins, this step is facilitated by Asn or Gln. For a Synechococcus sp. PCC7002 intein, the isolated third step of protein splicing is more efficient with its native Asn than with substitution to Gln. For a Trichodesmium erythraeum intein, its native Gln facilitates the third step as efficiently as with Asn. Despite these differences, the yield of splicing is not affected, suggesting that the third step is influenced by mechanism-linked conformational changes. A conserved catalytic His and the penultimate residue also play roles in promoting side-chain cyclization.
[Mh] Termos MeSH primário: Inteínas/genética
Processamento de Proteína
Synechococcus/genética
Trichodesmium/genética
[Mh] Termos MeSH secundário: Mutação
[Pt] Tipo de publicação:LETTER
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170815
[Lr] Data última revisão:
170815
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170627
[St] Status:MEDLINE
[do] DOI:10.1002/1873-3468.12730


  2 / 539 MEDLINE  
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[PMID]:28488863
[Au] Autor:Zwarycz AS; Fossat M; Akanyeti O; Lin Z; Rosenman DJ; Garcia AE; Royer CA; Mills KV; Wang C
[Ad] Endereço:Department of Biological Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
[Ti] Título:V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein.
[So] Source:Biochemistry;56(21):2715-2722, 2017 May 30.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini-mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this study, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔG °) of -4.4 and -6.9 kcal mol for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔV ) was significantly larger for V67 (71 ± 2 mL mol ) than for L67 (58 ± 3 mL mol ) inteins. The measured difference in ΔV (13 ± 3 mL mol ) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. It is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.
[Mh] Termos MeSH primário: Inteínas/genética
Leucina/genética
Mutação
Mycobacterium tuberculosis/enzimologia
Mycobacterium tuberculosis/genética
Recombinases Rec A/química
Recombinases Rec A/genética
Valina/genética
[Mh] Termos MeSH secundário: Fluorescência
Leucina/metabolismo
Simulação de Dinâmica Molecular
Ressonância Magnética Nuclear Biomolecular
Recombinases Rec A/metabolismo
Termodinâmica
Valina/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
EC 2.7.7.- (Rec A Recombinases); GMW67QNF9C (Leucine); HG18B9YRS7 (Valine)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170623
[Lr] Data última revisão:
170623
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170511
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.6b01264


  3 / 539 MEDLINE  
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[PMID]:28298556
[Au] Autor:Dougherty PG; Qian Z; Pei D
[Ad] Endereço:Department of Chemistry and Biochemistry, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, U.S.A.
[Ti] Título:Macrocycles as protein-protein interaction inhibitors.
[So] Source:Biochem J;474(7):1109-1125, 2017 Mar 15.
[Is] ISSN:1470-8728
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Macrocyclic compounds such as cyclic peptides have emerged as a new and exciting class of drug candidates for inhibition of intracellular protein-protein interactions, which are challenging targets for conventional drug modalities (i.e. small molecules and proteins). Over the past decade, several complementary technologies have been developed to synthesize macrocycle libraries and screen them for binding to therapeutically relevant targets. Two different approaches have also been explored to increase the membrane permeability of cyclic peptides. In this review, we discuss these methods and their applications in the discovery of macrocyclic compounds against protein-protein interactions.
[Mh] Termos MeSH primário: Biblioteca de Peptídeos
Peptídeos Cíclicos/farmacologia
Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos
Proteínas/antagonistas & inibidores
Bibliotecas de Moléculas Pequenas/farmacologia
[Mh] Termos MeSH secundário: Animais
Produtos Biológicos/síntese química
Produtos Biológicos/isolamento & purificação
Produtos Biológicos/farmacologia
Transporte Biológico
Permeabilidade da Membrana Celular/efeitos dos fármacos
Difusão
Descoberta de Drogas
Células Eucarióticas/citologia
Células Eucarióticas/efeitos dos fármacos
Células Eucarióticas/metabolismo
Seres Humanos
Inteínas/efeitos dos fármacos
Peptídeos Cíclicos/síntese química
Ligação Proteica/efeitos dos fármacos
Proteínas/química
Bibliotecas de Moléculas Pequenas/síntese química
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (Biological Products); 0 (Peptide Library); 0 (Peptides, Cyclic); 0 (Proteins); 0 (Small Molecule Libraries)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170615
[Lr] Data última revisão:
170615
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170317
[St] Status:MEDLINE
[do] DOI:10.1042/BCJ20160619


  4 / 539 MEDLINE  
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[PMID]:28258013
[Au] Autor:Tavassoli A
[Ad] Endereço:Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom. Electronic address: ali1@soton.ac.uk.
[Ti] Título:SICLOPPS cyclic peptide libraries in drug discovery.
[So] Source:Curr Opin Chem Biol;38:30-35, 2017 Jun.
[Is] ISSN:1879-0402
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Cyclic peptide libraries have demonstrated significant potential when employed against challenging targets such as protein-protein interactions. While a variety of methods for library generation exist, genetically encoded libraries hold several advantages over their chemically synthesized counterparts; they are more readily accessible and allow straightforward hit deconvolution. One method for the intracellular generation of such libraries is split-intein circular ligation of peptides and proteins (SICLOPPS). Here we detail and discuss the deployment of SICLOPPS libraries for the identification of cyclic peptide inhibitors of a variety of targets.
[Mh] Termos MeSH primário: Descoberta de Drogas/métodos
Inteínas
Biblioteca de Peptídeos
Peptídeos Cíclicos/química
[Mh] Termos MeSH secundário: Animais
Seres Humanos
Peptídeos Cíclicos/farmacologia
Proteínas/química
Proteínas/farmacologia
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (Peptide Library); 0 (Peptides, Cyclic); 0 (Proteins)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170719
[Lr] Data última revisão:
170719
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170305
[St] Status:MEDLINE


  5 / 539 MEDLINE  
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[PMID]:28165720
[Au] Autor:Minteer CJ; Siegart NM; Colelli KM; Liu X; Linhardt RJ; Wang C; Gomez AV; Reitter JN; Mills KV
[Ad] Endereço:Department of Chemistry, College of the Holy Cross , Worcester, Massachusetts 01610, United States.
[Ti] Título:Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage.
[So] Source:Biochemistry;56(8):1042-1050, 2017 Feb 28.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.
[Mh] Termos MeSH primário: Ácido Aspártico/metabolismo
DNA Polimerase II/química
Inteínas
[Mh] Termos MeSH secundário: Ácido Aspártico/química
Domínio Catalítico
Ciclização
DNA Polimerase II/genética
DNA Polimerase II/metabolismo
Temperatura Alta
Concentração de Íons de Hidrogênio
Mutação
Processamento de Proteína Pós-Traducional
Pyrococcus abyssi/enzimologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
30KYC7MIAI (Aspartic Acid); EC 2.7.7.- (DNA Polymerase II)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170516
[Lr] Data última revisão:
170516
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170207
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.6b00894


  6 / 539 MEDLINE  
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[PMID]:28100006
[Au] Autor:Shuai Z; Wang J; Badamagunta M; Choi J; Yang G; Zhang W; Kenny TP; Guggenheim K; Kurth MJ; Ansari AA; Voss J; Coppel RL; Invernizzi P; Leung PSC; Gershwin ME
[Ad] Endereço:Department of Rheumatology and Immunology, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
[Ti] Título:The fingerprint of antimitochondrial antibodies and the etiology of primary biliary cholangitis.
[So] Source:Hepatology;65(5):1670-1682, 2017 May.
[Is] ISSN:1527-3350
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The identification of environmental factors that lead to loss of tolerance has been coined the holy grail of autoimmunity. Our work has focused on the reactivity of antimitochondrial autoantibodies (AMA) to chemical xenobiotics and has hypothesized that a modified peptide within PDC-E2, the major mitochondrial autoantigen, will have been immunologically recognized at the time of loss of tolerance. Herein, we successfully applied intein technology to construct a PDC-E2 protein fragment containing amino acid residues 177-314 of PDC-E2 by joining a recombinant peptide spanning residues 177-252 (PDC-228) with a 62-residue synthetic peptide from 253 to 314 (PP), which encompasses PDC-E2 inner lipoyl domain (ILD). We named this intein-constructed fragment PPL. Importantly, PPL, as well as lipoic acid conjugated PPL (LA-PPL) and xenobiotic 2-octynoic acid conjugated PPL (2OA-PPL), are recognized by AMA. Of great importance, AMA has specificity for the 2OA-modified PDC-E2 ILD peptide backbone distinct from antibodies that react with native lipoylated PDC-E2 peptide. Interestingly, this unique AMA subfraction is of the immunoglobulin M isotype and more dominant in early-stage primary biliary cholangitis (PBC), suggesting that exposure to 2OA-PPL-like compounds occurs early in the generation of AMA. To understand the structural basis of this differential recognition, we analyzed PPL, LA-PPL, and 2OA-PPL using electron paramagnetic resonance spectroscopy, with confirmations by enzyme-linked immunosorbent assay, immunoblotting, and affinity antibody analysis. We demonstrate that the conformation of PDC-E2 ILD is altered when conjugated with 2OA, compared to conjugation with lipoic acid. CONCLUSION: A molecular understanding of the conformation of xenobiotic-modified PDC-E2 is critical for understanding xenobiotic modification and loss of tolerance in PBC with widespread implications for a role of environmental chemicals in the induction of autoimmunity. (Hepatology 2017;65:1670-1682).
[Mh] Termos MeSH primário: Autoanticorpos/sangue
Colangite/induzido quimicamente
Mitocôndrias/imunologia
Piruvato Desidrogenase (Lipoamida)/efeitos dos fármacos
Xenobióticos/toxicidade
[Mh] Termos MeSH secundário: Afinidade de Anticorpos
Estudos de Casos e Controles
Colangite/sangue
Colangite/imunologia
Espectroscopia de Ressonância de Spin Eletrônica
Ensaio de Imunoadsorção Enzimática
Seres Humanos
Inteínas
Piruvato Desidrogenase (Lipoamida)/química
Piruvato Desidrogenase (Lipoamida)/imunologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Autoantibodies); 0 (Xenobiotics); EC 1.2.4.1 (Pyruvate Dehydrogenase (Lipoamide))
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170523
[Lr] Data última revisão:
170523
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170119
[St] Status:MEDLINE
[do] DOI:10.1002/hep.29059


  7 / 539 MEDLINE  
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[PMID]:27881685
[Au] Autor:Shibuya Y; Haga N; Asano R; Nakazawa H; Hattori T; Takeda D; Sugiyama A; Kurotani R; Kumagai I; Umetsu M; Makabe K
[Ad] Endereço:Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa, Yamagata 992-8510, Japan.
[Ti] Título:Generation of camelid VHH bispecific constructs via in-cell intein-mediated protein trans-splicing.
[So] Source:Protein Eng Des Sel;30(1):15-21, 2017 Jan.
[Is] ISSN:1741-0134
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Production of various combinations of bispecific variable domain of heavy chain of heavy chain-only antibody (VHH) constructs to evaluate their therapeutic potential usually requires several gene-engineering steps. Here, we present an alternative method of creating bispecific VHH constructs in vivo through protein trans-splicing (PTS) reaction; this method may reduce the number of gene manipulation steps required. As a proof-of-concept, we constructed a bispecific antibody (bsAb) containing an anti-epidermal growth factor receptor VHH and anti-green fluorescent protein VHH, and we evaluated and confirmed its bispecificity. We also tested antibody labeling by fluorescent protein tagging using the PTS reaction. Compared with the conventional gene construction method, bsAb construction via PTS is a promising alternative approach for generating multiple bsAb combinations.
[Mh] Termos MeSH primário: Anticorpos Biespecíficos/química
Anticorpos Biespecíficos/genética
Inteínas
Engenharia de Proteínas
Anticorpos de Domínio Único/química
Anticorpos de Domínio Único/genética
Trans-Splicing
[Mh] Termos MeSH secundário: Animais
Anticorpos Biespecíficos/imunologia
Células CHO
Camelídeos Americanos
Cricetulus
Seres Humanos
Anticorpos de Domínio Único/imunologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antibodies, Bispecific); 0 (Single-Domain Antibodies)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170503
[Lr] Data última revisão:
170503
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161125
[St] Status:MEDLINE


  8 / 539 MEDLINE  
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[PMID]:27852847
[Au] Autor:Zhao F; Zhao T; Deng L; Lv D; Zhang X; Pan X; Xu J; Long G
[Ad] Endereço:Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
[Ti] Título:Visualizing the Essential Role of Complete Virion Assembly Machinery in Efficient Hepatitis C Virus Cell-to-Cell Transmission by a Viral Infection-Activated Split-Intein-Mediated Reporter System.
[So] Source:J Virol;91(2), 2017 Jan 15.
[Is] ISSN:1098-5514
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Hepatitis C virus (HCV) infects 2 to 3% of the world population and is a leading cause of liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. Many aspects of HCV study, ranging from molecular virology and antiviral drug development to drug resistance profiling, were supported by straightforward assays of HCV replication and infection. Among these assays, the HCV-dependent fluorescence relocalization (HDFR) system allowed live-cell visualization of infection without modifying the viral genome, but this strategy required careful recognition of the fluorescence relocalization pattern for its high fluorescence background in the cytoplasm. In this study, to achieve background-free visualization of HCV infection, a viral infection-activated split-intein-mediated reporter system (VISI) was devised. Uninfected Huh7.5.1-VISI cells show no background signal, while HCV infection specifically illuminates the nuclei of infected Huh7.5.1-VISI cells with either green fluorescent protein (GFP) or mCherry. Combining VISI-GFP and VISI-mCherry systems, we revisited HCV cell-to-cell transmission with clear-cut distinction of donor and recipient cells in a live-cell manner. Independently of virion assembly, exosomes have been reported to transfer HCV subgenomic RNA to initiate replication in uninfected cells, which suggested an assembly-free pathway. However, our data demonstrated that HCV structural genes and the p7 gene were essential for not only cell-free infectivity but also cell-to-cell transmission. Additionally, depletion of apolipoprotein E (ApoE) from donor cells but not from recipient cells significantly reduced HCV cell-to-cell transmission efficiency. In summary, we developed a background-free cell-based reporter system for convenient live-cell visualization of HCV infection, and our data indicate that complete HCV virion assembly machinery is essential for both cell-free and cell-to-cell transmission. IMPORTANCE: Hepatitis C virus (HCV) infects hepatocytes via two pathways: cell-free infection and cell-to-cell transmission. Structural modules of the HCV genome are required for production of infectious cell-free virions; however, the role of specific genes within the structural module in cell-to-cell transmission is not clearly defined. Our data demonstrate that deletion of core, E1E2, and p7 genes individually results in no HCV cell-to-cell transmission and that ApoE knockdown from donor cells causes less-efficient cell-to-cell transmission. Thus, this work indicates that the complete HCV assembly machinery is required for HCV cell-to-cell transmission. At last, this work presents an optimized viral infection-activated split-intein-mediated reporter system for easy live-cell monitoring of HCV infection.
[Mh] Termos MeSH primário: Hepacivirus/fisiologia
Hepatite C/metabolismo
Hepatite C/virologia
Inteínas
Receptores Virais
Montagem de Vírus
Replicação Viral
[Mh] Termos MeSH secundário: Antivirais/farmacologia
Apolipoproteínas E/metabolismo
Linhagem Celular
Células Cultivadas
Expressão Gênica
Ordem dos Genes
Genes Reporter
Vetores Genéticos/genética
Hepacivirus/efeitos dos fármacos
Interações Hospedeiro-Patógeno
Seres Humanos
Proteínas Virais/genética
Proteínas Virais/metabolismo
Vírion
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antiviral Agents); 0 (Apolipoproteins E); 0 (Receptors, Virus); 0 (Viral Proteins); 0 (p7 protein, Hepatitis C virus)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170714
[Lr] Data última revisão:
170714
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161118
[St] Status:MEDLINE


  9 / 539 MEDLINE  
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[PMID]:27704298
[Au] Autor:Tori K; Perler F
[Ad] Endereço:New England Biolabs, Inc., Ipswich, MA, 01938, USA.
[Ti] Título:Sequential formation of two branched intermediates during protein splicing of class three inteins.
[So] Source:Extremophiles;21(1):41-49, 2017 Jan.
[Is] ISSN:1433-4909
[Cp] País de publicação:Germany
[La] Idioma:eng
[Ab] Resumo:Inteins are the protein equivalent of introns. They are seamlessly removed during post-translational maturation of their host protein (extein). Inteins from extremophiles played a key role in understanding intein-mediated protein splicing. There are currently three classes of inteins defined by catalytic mechanism and sequence signatures. This study demonstrates splicing of three class 3 mini-inteins: Burkholderia vietnamiensis G4 Bvi IcmO intein, Mycobacterium smegmatis MC2 155 Msm DnaB-1 intein and Mycobacterium leprae strain TN Mle DnaB intein. B. vietnamiensis has a broad ecological range and remediates trichloroethene. M. smegmatis is a biofilm forming soil bacteria. Although other intein classes have only a single branched intermediate at the C-terminal splice junction, the class 3 intein reaction pathway includes two branched intermediates. The class 3 specific branched intermediate is formed by an internal cysteine, while the C-terminal branch intermediate is at a serine or threonine in all class 3 inteins except the Bvi IcmO intein, where it is a cysteine. This latter cysteine was unable to compensate for mutation of the class 3-specific internal catalytic cysteine despite the Bvi IcmO intein having an N-terminal splice junction naturally tuned for a cysteine nucleophile, demonstrating the mandatory order of branch intermediates in class 3 inteins.
[Mh] Termos MeSH primário: Proteínas de Bactérias/metabolismo
Burkholderia/metabolismo
Inteínas
Mycobacterium leprae/metabolismo
Processamento de Proteína Pós-Traducional
[Mh] Termos MeSH secundário: Proteínas de Bactérias/química
Burkholderia/genética
Mycobacterium leprae/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins)
[Em] Mês de entrada:1702
[Cu] Atualização por classe:170223
[Lr] Data última revisão:
170223
[Sb] Subgrupo de revista:IM; S
[Da] Data de entrada para processamento:161006
[St] Status:MEDLINE
[do] DOI:10.1007/s00792-016-0876-0


  10 / 539 MEDLINE  
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[PMID]:27683210
[Au] Autor:Shi S; Chen H; Jiang H; Xie Y; Zhang L; Li N; Zhu C; Chen J; Luo H; Wang J; Feng L; Lu H; Zhu J
[Ad] Endereço:Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
[Ti] Título:A novel self-cleavable tag Zbasic-∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli.
[So] Source:Appl Microbiol Biotechnol;101(3):1133-1142, 2017 Feb.
[Is] ISSN:1432-0614
[Cp] País de publicação:Germany
[La] Idioma:eng
[Ab] Resumo:Soluble expression of recombinant therapeutic proteins in Escherichia coli (E. coli) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic-intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable Mycobacterium tuberculosis recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein "Zbasic-∆I-CM-IL-15" was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic-∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic-∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in E. coli.
[Mh] Termos MeSH primário: Escherichia coli/genética
Interleucina-15/química
Interleucina-15/genética
[Mh] Termos MeSH secundário: Biofarmácia/métodos
Cromatografia Líquida
Escherichia coli/metabolismo
Seres Humanos
Concentração de Íons de Hidrogênio
Inteínas
Interleucina-15/isolamento & purificação
Espectrometria de Massas
Mycobacterium tuberculosis/genética
Proteínas Recombinantes de Fusão/química
Proteínas Recombinantes de Fusão/economia
Proteínas Recombinantes de Fusão/isolamento & purificação
Solubilidade
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (IL15 protein, human); 0 (Interleukin-15); 0 (Recombinant Fusion Proteins)
[Em] Mês de entrada:1701
[Cu] Atualização por classe:170130
[Lr] Data última revisão:
170130
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160930
[St] Status:MEDLINE
[do] DOI:10.1007/s00253-016-7848-2



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