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1.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
2.
Cancer Sci ; 110(9): 2722-2733, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31461572

RESUMO

Mesothelin (MSLN) shows increased expression in various cancer cells. For clinical application of antibodies as a positron emission tomography (PET) imaging reagent, a human shortened antibody is essential both for avoiding redundant immune responses and for providing rapid imaging. Therefore, we cloned a single-chain fragment of variable regions (scFv) from a human-derived gene sequence. This was achieved through the construction of a naïve phage library derived from human tonsil lymphocytes. Using a column with human recombinant MSLN, we carried out bio-panning of phage-variants by colony formation. We first obtained 120 clones that were subjected to selection in an ELISA using human recombinant MSLN as a solid phase antigen, and 15 phage clones of scFv with a different sequence were selected and investigated by flow cytometry (FCM). Then, six variants were selected and the individual scFv gene was synthesized in the VL and VH domains and expressed in Chinese hamster ovary cells. Mammalian cell-derived human-origin scFv clones were analyzed by FCM again, and one MSLN highly specific scFv clone was established. PET imaging by 89 Zr-labeled scFv was done in mice bearing xenografts with MSLN-expressing cancer cells, and tumor legions were successfully visualized. The scFv variant established in the present study may be potentially useful for cancer diagnosis by PET imaging.


Assuntos
Proteínas Ligadas por GPI/imunologia , Neoplasias/diagnóstico por imagem , Compostos Radiofarmacêuticos/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Células CHO , Linhagem Celular Tumoral , Clonagem Molecular , Cricetulus , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/isolamento & purificação , Proteínas Ligadas por GPI/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Imagem Molecular/métodos , Neoplasias/patologia , Biblioteca de Peptídeos , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons/métodos , Radioisótopos , Compostos Radiofarmacêuticos/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , Zircônio
3.
BMC Infect Dis ; 19(1): 622, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307416

RESUMO

BACKGROUND: Cell-surface mucins are expressed in apical epithelial cells of the respiratory tract, and contribute a crucial part of the innate immune system. Despite anti-inflammatory or antiviral functions being revealed for certain cell-surface mucins such as MUC1, the roles of other mucins are still poorly understood, especially in viral infections. METHODS: To further identify mucins significant in influenza infection, we screened the expression of mucins in human nasal epithelial cells infected by H3N2 influenza A virus. RESULTS: We found that the expression of MUC15 was significantly upregulated upon infection, and specific only to active infection. While MUC15 did not interact with virus particles or reduce viral replication directly, positive correlations were observed between MUC15 and inflammatory factors in response to viral infection. Given that the upregulation of MUC15 was only triggered late into infection when immune factors (including cytokines, chemokines, EGFR and phosphorylated ERK) started to peak and plateau, MUC15 may potentially serve an immunomodulatory function later during influenza viral infection. CONCLUSIONS: Our study revealed that MUC15 was one of the few cell-surface mucins induced during influenza infection. While MUC15 did not interact directly with influenza virus, we showed that its increase coincides with the peak of immune activation and thus MUC15 may serve an immunomodulatory role during influenza infection.


Assuntos
Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Humana/patologia , Mucinas/metabolismo , Animais , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Cães , Células Epiteliais/classificação , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Humanos , Influenza Humana/metabolismo , Células Madin Darby de Rim Canino , Mucinas/antagonistas & inibidores , Mucinas/genética , Cavidade Nasal/citologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Regulação para Cima , Replicação Viral/efeitos dos fármacos
4.
J Chromatogr A ; 1602: 341-349, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31204039

RESUMO

Protein purification by affinity chromatography relies primarily on the interaction of a fused-tag to the protein of interest. Here, we describe a tag-free affinity method that employs functional selection interactions to a broad range of proteins. To achieve this, we coupled human DNA-clamp proliferating cell nuclear antigen (PCNA) that interacts with over one hundred proteins to an agarose resin. We demonstrate the versatility of our PCNA-Agarose column at various chromatographic steps by purifying PCNA-binding proteins that are involved in DNA Replication (DNA polymerase δ, flap endonuclease 1 and DNA ligase 1), translesion DNA synthesis (DNA polymerases eta, kappa and iota) and genome stability (p15). We also show the competence of the PCNA-Agarose column to purify non-PCNA binding proteins by fusing the PCNA-binding motif of human p21 as an affinity tag. Finally, we establish that our PCNA-Agarose column is a suitable analytical method for characterizing the binding strength of PCNA-binding proteins. The conservation and homology of PCNA-like clamps will allow for the immediate extension of our method to other species.


Assuntos
Cromatografia de Afinidade/métodos , Antígeno Nuclear de Célula em Proliferação/isolamento & purificação , Sefarose/química , Tampões (Química) , DNA Polimerase III/isolamento & purificação , Reparo do DNA , Replicação do DNA , Humanos , Ligação Proteica , Proteínas Recombinantes/isolamento & purificação , Resinas Sintéticas/química
5.
Food Chem ; 297: 124925, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253266

RESUMO

A novel lipase gene (McLipB) was cloned from a thermophilic fungus Malbranchea cinnamomea and expressed in Pichia pastoris. The deduced amino acid sequence of the lipase (McLipB) shared the highest identity of 46% with the Candida rugosa lipase LIP4. The extracellular lipase activity of 4304 U/mL with protein concentration of 7.7 mg/mL was achieved in a 5-L fermentor. The optimal pH and temperature of McLipB were 7.5 and 40 °C, respectively. The lipase showed high specificity towards triglycerides with short and medium chain fatty acids, and had non-position specificity. McLipB hydrolyzed butter to produce mainly butyric acid, hexanoic acid and a small amount of octanoic acid and decanoic acid. Furthermore, it degraded more than 90% dipropyl phthalate, dibutyl phthalate and dihexyl phthalate to their corresponding monoalkyl phthalates. The properties of McLipB indicate that it has great application potential for production of lipolyzed milkfat flavor and biodegradation of phthalate esters.


Assuntos
Ascomicetos/enzimologia , Lipase/metabolismo , Sequência de Aminoácidos , Técnicas de Cultura Celular por Lotes , Candida/enzimologia , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/química , Lipase/genética , Ácidos Ftálicos/química , Ácidos Ftálicos/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Especificidade por Substrato , Temperatura Ambiente , Triglicerídeos/metabolismo
6.
Prep Biochem Biotechnol ; 49(8): 813-821, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169457

RESUMO

Separation of biomass from culture media by centrifugation and then washing the biomass are mandatory steps in the fermentation process of recombinant Pichia pastoris expressed HBsAg intracellularly. Biomass has to be washed many times to eliminate the culture media residues thoroughly. In this study, we tried to develop the hydrocyclone as an alternative method for separation of biomass from fermentation culture, an attractive replacement for centrifugation processes. The advantages of using hydrocyclone in biomass separation could be summarized in its suitability for continuous separation and its low risk of contamination. To evaluate the performance of hydrocyclone, concentration ratio in underflow to feed stream, capacity, and centrifugal force by considering three parameters of pressure drop, concentration, and the type of hydrocyclone were investigated. Using three level factorial design a concentration ratio equation was developed, with the correlation coefficient R2 = 0.977 ensured the good fitness of the predicted data with the experimental results. In optimal conditions, maximum concentration ratio was 1.246, for flow rate 13.5 LPM and C-force equal to 1276.11 at maximum pressure drop (3 bar) and minimum concentration (0.5% w/w) in hydrocyclone 1. Herein, two different hydrocyclones with the cylindrical diameters of 19 mm and 21 mm were used for separating the yeast cells.


Assuntos
Centrifugação/instrumentação , Meios de Cultura/química , Antígenos de Superfície da Hepatite B/isolamento & purificação , Pichia/química , Técnicas de Cultura Celular por Lotes/instrumentação , Biomassa , Desenho de Equipamento , Fermentação , Pressão , Proteínas Recombinantes/isolamento & purificação
7.
Nat Commun ; 10(1): 2636, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201318

RESUMO

The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Modelos Moleculares , Cristalografia por Raios X , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Anticorpos de Domínio Único/metabolismo
8.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
9.
Nat Commun ; 10(1): 2649, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201333

RESUMO

In human and other mammalian cells, transport of L-lactate across plasma membranes is mainly catalyzed by monocarboxylate transporters (MCTs) of the SLC16 solute carrier family. MCTs play an important role in cancer metabolism and are promising targets for tumor treatment. Here, we report the crystal structures of an SLC16 family homologue with two different bound ligands at 2.54 and 2.69 Å resolution. The structures show the transporter in the pharmacologically relevant outward-open conformation. Structural information together with a detailed structure-based analysis of the transport function provide important insights into the molecular working mechanisms of ligand binding and L-lactate transport.


Assuntos
Proteínas de Bactérias/química , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Transporte de Íons/fisiologia , Ligantes , Transportadores de Ácidos Monocarboxílicos/isolamento & purificação , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/química , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Simportadores/química
10.
Nat Commun ; 10(1): 2693, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217419

RESUMO

The kinesin-3 KIF1C is a fast organelle transporter implicated in the transport of dense core vesicles in neurons and the delivery of integrins to cell adhesions. Here we report the mechanisms of autoinhibition and release that control the activity of KIF1C. We show that the microtubule binding surface of KIF1C motor domain interacts with its stalk and that these autoinhibitory interactions are released upon binding of protein tyrosine phosphatase PTPN21. The FERM domain of PTPN21 stimulates dense core vesicle transport in primary hippocampal neurons and rescues integrin trafficking in KIF1C-depleted cells. In vitro, human full-length KIF1C is a processive, plus-end directed motor. Its landing rate onto microtubules increases in the presence of either PTPN21 FERM domain or the cargo adapter Hook3 that binds the same region of KIF1C tail. This autoinhibition release mechanism allows cargo-activated transport and might enable motors to participate in bidirectional cargo transport without undertaking a tug-of-war.


Assuntos
Cinesina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Vesículas Citoplasmáticas/metabolismo , Hipocampo/citologia , Humanos , Integrinas/metabolismo , Microscopia Intravital/métodos , Cinesina/genética , Cinesina/isolamento & purificação , Camundongos , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Microtúbulos/metabolismo , Neurônios/citologia , Cultura Primária de Células , Ligação Proteica , Domínios Proteicos , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/isolamento & purificação , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/métodos
11.
Nat Commun ; 10(1): 2714, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221956

RESUMO

The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Proteínas de Drosophila/metabolismo , Simulação de Dinâmica Molecular , Sódio/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/isolamento & purificação , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Células HEK293 , Humanos , Ligantes , Espectrometria de Massas , Conformação Proteica em alfa-Hélice/fisiologia , Domínios Proteicos/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
12.
Nat Commun ; 10(1): 2775, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235796

RESUMO

The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization.


Assuntos
Biocatálise , Endopeptidases/metabolismo , Proteínas de Plantas/metabolismo , Poliproteínas/síntese química , Engenharia de Proteínas/métodos , Estudos de Viabilidade , Microscopia de Força Atômica/métodos , Oldenlandia , Poliproteínas/genética , Poliproteínas/isolamento & purificação , Poliproteínas/ultraestrutura , Potyvirus , Desdobramento de Proteína , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Rubredoxinas/síntese química , Rubredoxinas/genética , Rubredoxinas/isolamento & purificação , Rubredoxinas/ultraestrutura , Imagem Individual de Molécula/métodos , Análise Espectral/métodos , Proteínas Virais
13.
Biochemistry (Mosc) ; 84(4): 390-397, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228930

RESUMO

Recombinant malic enzyme from the aerobic methanotroph Methylosinus trichosporium was obtained by heterologous expression in Escherichia coli and purified by affinity metal-chelating chromatography. The homohexameric enzyme of 6×80 kDa catalyzed the reversible reaction of oxidative decarboxylation of malate to pyruvate in the presence of mono- and divalent cations and NADP+ as a cofactor. The kcat/Km ratio indicated much higher catalytic efficiency of the malate decarboxylation reaction as compared with the pyruvate carboxylation reaction. Analysis of the protein sequence revealed that the C-region of the enzyme contains a large domain homologous to phosphoacetyltransferase, but no phosphoacetyltransferase activity was detected either for a full chimeric malic enzyme or for the C-end fragment obtained as a separate protein. This C-end domain promoted activity of the malic enzyme.


Assuntos
Malato Desidrogenase/metabolismo , Methylosinus trichosporium/enzimologia , Biocatálise , Descarboxilação , Cinética , Malato Desidrogenase/química , Malato Desidrogenase/genética , NADP/metabolismo , Ácido Pirúvico/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
14.
Biochemistry (Mosc) ; 84(4): 426-434, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228934

RESUMO

The bacterium Escherichia coli has seven σ subunits that bind core RNA polymerase and are necessary for promoter recognition. It was previously shown that the σ70 and σ38 subunits can also interact with the transcription elongation complex (TEC) and stimulate pausing by recognizing DNA sequences similar to the -10 element of promoters. In this study, we analyzed the ability of the σ32, σ28, and σ24 subunits to induce pauses in reconstituted TECs containing corresponding -10 consensus elements. It was found that the σ24 subunit can induce a transcriptional pause depending on the presence of the -10 element. Pause formation is suppressed by the Gre factors, suggesting that the paused complex adopts a backtracked conformation. Some natural promoters contain potential signals of σ24-dependent pauses in the initially transcribed regions, suggesting that such pauses may have regulatory functions in transcription.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Transcrição Genética/fisiologia , Sequência de Bases , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo
15.
Food Chem ; 295: 653-661, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174809

RESUMO

Although ß-xylosidases have a wide range of applications, cold-active ß-xylosidases have been poorly studied. In this study, a cold active ß-xylosidase gene (xyl) from Bacillus pumilus TCCC 11,350 was cloned and overexpressed in Escherichia coli. The recombinant XYL (rXYL) was revealed to be a bifunctional enzyme with both ß-xylosidase and α-l-arabinofuranosidase activities. Purified rXYL was most active at 30 °C, demonstrating 26% and 18% of its maximum activity at 4 °C and 0 °C, respectively. Meanwhile, rXYL showed a 52% activity in 200 mM xylose, indicating a relatively strong tolerance to xylose. Moreover, rXYL exhibited a high synergistic effect (11.14-fold and 16.21-fold) with endo-xylanase to degrade beechwood xylan in both sequential and simultaneous reactions at low temperatures. As the first report on the novel cold-adapted ß-xylosidase from B. pumilus, these results suggested rXYL had attractive properties for food industrial utilizations.


Assuntos
Bacillus pumilus/enzimologia , Xilosidases/metabolismo , Sequência de Aminoácidos , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Temperatura Ambiente , Xilanos/metabolismo , Xilose/metabolismo , Xilosidases/classificação , Xilosidases/genética
16.
Chem Biol Interact ; 309: 108698, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31176713

RESUMO

Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 Å resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature-related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under conditions approaching physiological temperature.


Assuntos
Acetilcolinesterase/química , Cristalografia por Raios X , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Aminacrina/química , Aminacrina/metabolismo , Sítios de Ligação , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Dimerização , Humanos , Simulação de Dinâmica Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura Ambiente
17.
Biochemistry (Mosc) ; 84(5): 520-528, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234766

RESUMO

In our recent X-ray study, we demonstrated that substitution of the natural leucine residue M196 with histidine in the reaction center (RC) from Rhodobacter (Rba.) sphaeroides leads to formation of a close contact between the genetically introduced histidine and the primary electron donor P (bacteriochlorophylls (BChls) PA and PB dimer) creating a novel pigment-protein interaction that is not observed in native RCs. In the present work, the possible nature of this novel interaction and its effects on the electronic properties of P and the photochemical charge separation in isolated mutant RCs L(M196)H are investigated at room temperature using steady-state absorption spectroscopy, light-induced difference FTIR spectroscopy, and femtosecond transient absorption spectroscopy. The results are compared with the data obtained for the RCs from Rba. sphaeroides pseudo-wild type strain. It is shown that the L(M196)H mutation results in a decrease in intensity and broadening of the long-wavelength Qy absorption band of P at ~865 nm. Due to the mutation, there is also weakening of the electronic coupling between BChls in the radical cation P+ and increase in the positive charge localization on the PA molecule. Despite the significant perturbations of the electronic structure of P, the mutant RCs retain high electron transfer rates and quantum yield of the P+QA- state (QA is the primary quinone acceptor), which is close to the one observed in the native RCs. Comparison of our results with the literature data suggests that the imidazole group of histidine M196 forms a π-hydrogen bond with the π-electron system of the PB molecule in the P dimer. It is likely that the specific (T-shaped) spatial organization of the π-hydrogen interaction and its potential heterogeneity in relation to the bonding energy is, at least partially, the reason that this type of interaction between the protein and the pigment and quinone cofactors is not realized in the native RCs.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina/metabolismo , Leucina/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Transporte de Elétrons , Histidina/genética , Cinética , Leucina/genética , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier
18.
Biochemistry (Mosc) ; 84(5): 540-552, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234768

RESUMO

L-Arabinose is an important component of mycobacterial cell wall. L-Arabinose is involved in the synthesis of arabinogalactan, lipoarabinomannan, and other sugar compounds, which suggests that it can modulate cell wall permeability and drug resistance. However, whether L-arabinose affects mycobacterial antibiotic resistance and the underlying regulatory mechanism remains unclear. In this study, we characterized a new transcription factor of Mycobacterium smegmatis, AraR, that responds to L-arabinose and regulates mycobacterial sensitivity to isoniazid (INH). AraR specifically recognizes two conserved 15-bp motifs within the upstream regulatory region of the arabinose (araR) operon. AraR functions as a transcriptional repressor that negatively regulates araR expression. In contrast to the effect of AraR, overexpression of the araR operon contributes to the mycobacterial INH resistance. L-arabinose acts as an effector and derepresses transcriptional inhibition by AraR. The araR-deficient strain is more resistant to INH than the wild-type strain, whereas the araR-overexpressing strain is more sensitive to INH. Addition of L-arabinose to the medium can significantly increase the resistance to INH of the wild-type strain, but not of the araR knockout strain. Therefore, we identified a new L-arabinose-responding transcription factor and revealed its effect on the bacterial antibiotic resistance. These findings can provide new insights in the regulatory mechanisms mediated by sugar molecules and their relationship with drug resistance in mycobacteria.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Isoniazida/farmacologia , Mycobacterium smegmatis/metabolismo , Fatores de Transcrição/metabolismo , Arabinose/química , Arabinose/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Mycobacterium smegmatis/efeitos dos fármacos , Óperon , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Fatores de Transcrição/genética
19.
Biochemistry (Mosc) ; 84(6): 627-636, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238862

RESUMO

The cytokine TRAIL induces apoptosis in tumor cells of various origin without affecting normal cells. Clinical trials of TRAIL-receptor (DR4 and DR5) agonists (recombinant TRAIL or death receptors antibodies) have largely failed because most human tumors were resistant to them. Currently, a second generation of agents targeted at TRAIL-R with increased efficiency has been developed. To this end, we have developed DR5-B, a variant of TRAIL selectively interacting with DR5. We have developed a new efficient method for production of TRAIL and DR5-B using expression of these proteins in Escherichia coli strain SHuffle B. The proteins were isolated from the cytoplasmic fraction of cells and purified to a high degree of homogeneity using metal-affinity and ion-exchange chromatography. The protein yield was 211 and 173 mg from one liter of cell culture for DR5-B and TRAIL, respectively, which significantly exceeded the results obtained by other methods. DR5-B killed tumor cells of different origin more efficiently and rapidly compared with TRAIL. The resulting preparations can be used for the study of TRAIL signaling pathways and in preclinical and clinical trials as antitumor agents.


Assuntos
Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia
20.
Nat Commun ; 10(1): 2015, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043610

RESUMO

Previous studies have utilized monoamine oxidase (MAO) and L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces (R,S)-THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.


Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Escherichia coli/metabolismo , Proteínas de Insetos/metabolismo , Engenharia Metabólica/métodos , Tetra-Hidropapaverolina/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Motivos de Aminoácidos/genética , Animais , Descarboxilases de Aminoácido-L-Aromático/química , Descarboxilases de Aminoácido-L-Aromático/genética , Descarboxilases de Aminoácido-L-Aromático/isolamento & purificação , Bombyx , Dopamina/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
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