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1.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 324-331, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045561

RESUMO

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.


Assuntos
Proteínas de Bactérias/química , Hidrocarbonetos Halogenados/química , Hidrolases/química , Psychrobacter/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Temperatura Baixa , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hidrocarbonetos Halogenados/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Psychrobacter/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica
2.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 332-339, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045562

RESUMO

SUMOylation is a post-translational modification in which a small ubiquitin-like molecule (SUMO) is appended to substrate proteins and is known to influence myriads of biological processes. A delicate interplay between several families of SUMOylation proteins and their substrates ensures the proper level of SUMOylation required for normal cell function. Among the SUMO proteins, SUMO2 is known to form mono-SUMOylated proteins and engage in poly-SUMO chain formation, while sentrin-specific protease 1 (SENP1) is a key enzyme in regulating both events. Determination of the SENP1-SUMO2 interaction is therefore necessary to better understand SUMOylation. In this regard, the current paper reports the noncovalent structure of SENP1 in complex with SUMO2, which was refined to a resolution of 2.62 Šwith R and Rfree values of 22.92% and 27.66%, respectively. The structure shows that SENP1-SUMO2 complex formation is driven largely by polar interactions and limited hydrophobic contacts. The essential C-terminal motif (QQTGG) of SUMO2 is stabilized by a number of specific bonding interactions that enable it to protrude into the catalytic triad of SENP1 and provide the arrangement necessary for maturation of SUMO and deSUMOylation activity. Overall, the structure shows a number of structural details that pinpoint the basis of SENP1-SUMO2 complex formation.


Assuntos
Cisteína Endopeptidases/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Homologia Estrutural de Proteína , Termodinâmica
3.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 340-347, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045563

RESUMO

Ebola virus is an emerging virus that is capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome are carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 Šresolution single-particle cryo-electron microscopy structure of the nucleoprotein-RNA helical filament presented here resembles previous structures determined at lower resolution, while providing improved molecular details of protein-protein and protein-RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid.


Assuntos
Ebolavirus/química , Nucleocapsídeo/química , Nucleoproteínas/química , RNA Viral/química , Proteínas Virais/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleocapsídeo/ultraestrutura , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Proteínas Virais/metabolismo
4.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 348-358, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045564

RESUMO

Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. The human POTs PepT1 and PepT2 are also involved in the absorption of various orally ingested drugs. Previously reported structures revealed that the bacterial POTs possess 14 helices, of which H1-H6 and H7-H12 constitute the typical MFS fold and the residual two helices are involved in the cytoplasmic linker. PepTSo2 from Shewanella oneidensis is a unique POT which reportedly assembles as a 200 kDa tetramer. Although the previously reported structures suggested the importance of H12 for tetramer formation, the structural basis for the PepTSo2-specific oligomerization remains unclear owing to the lack of a high-resolution tetrameric structure. In this study, the expression and purification conditions for tetrameric PepTSo2 were optimized. A single-particle cryo-EM analysis revealed the tetrameric structure of PepTSo2 incorporated into Salipro nanoparticles at 4.1 Šresolution. Furthermore, a combination of lipidic cubic phase (LCP) crystallization and an automated data-processing system for multiple microcrystals enabled crystal structures of PepTSo2 to be determined at resolutions of 3.5 and 3.9 Å. The present structures in a lipid bilayer revealed the detailed mechanism for the tetrameric assembly of PepTSo2, in which a characteristic extracellular loop (ECL) interacts with two asparagine residues on H12 which were reported to be important for tetramerization and plays an essential role in oligomeric assembly. This study provides valuable insights into the oligomerization mechanism of this MFS-type transporter, which will further pave the way for understanding other oligomeric membrane proteins.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Shewanella/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Shewanella/metabolismo , Especificidade por Substrato
5.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 359-367, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045565

RESUMO

As of 2017, tuberculosis had infected 1.7 billion people (23% of the population of the world) and caused ten million deaths. Mycobacterium tuberculosis (Mtb) is quickly evolving, and new strains are classified as multidrug resistant. Thus, the identification of novel druggable targets is essential to combat the proliferation of these drug-resistant strains. Filamenting temperature-sensitive mutant Z (FtsZ) is a key protein involved in cytokinesis, an important process for Mtb proliferation and viability. FtsZ is required for bacterial cell division because it polymerizes into a structure called the Z-ring, which recruits accessory division proteins to the septum. Here, the crystal structure of the MtbFtsZ protein has been determined to 3.46 Šresolution and is described as a dimer of trimers, with an inter-subunit interface between protomers AB and DE. In this work, a novel conformation of MtbFtsZ is revealed involving the T9 loop and the nucleotide-binding pocket of protomers BC and EF.


Assuntos
Proteínas de Bactérias/química , Proteínas do Citoesqueleto/química , Mycobacterium tuberculosis/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Divisão Celular , Clonagem Molecular , Cristalografia por Raios X , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Temperatura Ambiente
6.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 368-376, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045566

RESUMO

The bacterial periplasmic protein LpoA is an outer membrane lipoprotein and an activator for the cross-linking activity of PBP1A, a bifunctional peptidoglycan synthase. Previous structures of the amino-terminal (N) domain of LpoA showed it to consist entirely of helices and loops, with at least four tetratricopeptide-like repeats. Although the previously determined orthorhombic crystal structure of the N domain of Haemophilus influenzae LpoA showed a typical curved structure with a concave groove, an NMR structure of the same domain from Escherichia coli was relatively flat. Here, a crystal structure of the N domain of E. coli LpoA was determined to a resolution of 2.1 Šand was found to be more similar to the H. influenzae crystal structure than to the E. coli NMR structure. To provide a quantitative description for these comparisons, the various structures were superimposed pairwise by fitting the first half of each structure to its pairwise partner and then calculating the rotation axis that would optimally superimpose the second half. Differences in both the magnitude of the rotation and the direction of the rotation axis were observed between different pairs of structures. A 1.35 Šresolution structure of a monoclinic crystal form of the N domain of H. influenzae LpoA was also determined. In this structure, the subdomains rotate 10° relative to those in the original orthorhombic H. influenzae crystal structure to further narrow the groove between the subdomains. To accommodate this, a bound chloride ion (in place of sulfate) allowed the closer approach of a helix that forms one side of the groove.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Cloretos/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Haemophilus influenzae/química , Lipoproteínas/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína
7.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 377-384, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045567

RESUMO

With better tools for data processing and with synchrotron beamlines that are capable of collecting data at longer wavelengths, sulfur-based native single-wavelength anomalous dispersion (SAD) phasing has become the `first-choice' method for de novo protein structure determination. However, for many proteins native SAD phasing can be simplified by taking advantage of their interactions with natural metal cofactors that are stronger anomalous scatterers than sulfur. This is demonstrated here for four unique domains of a 1.5 MDa calcium-dependent adhesion protein using the anomalous diffraction of the chelated calcium ions. In all cases, low anomalous multiplicity X-ray data were collected on a home-source diffractometer equipped with a chromium rotating anode (λ = 2.2909 Å). In all but one case, calcium SAD phasing alone was sufficient to allow automated model building and refinement of the protein model after the calcium substructure had been determined. Given that Ca atoms will be present in a significant percentage of proteins that remain uncharacterized, many aspects of the data-collection and processing methods described here could be broadly applied for routine de novo structure elucidation.


Assuntos
Adesinas Bacterianas/química , Proteínas de Bactérias/química , Cálcio/química , Gelo/análise , Marinomonas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Organismos Aquáticos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cátions Bivalentes , Clonagem Molecular , Temperatura Baixa , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difração de Raios X
8.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 385-391, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045568

RESUMO

The inhibition of kallikrein 5 (KLK5) has been identified as a potential strategy for treatment of the genetic skin disorder Netherton syndrome, in which loss-of-function mutations in the SPINK5 gene lead to down-regulation of the endogenous inhibitor LEKTI-1 and profound skin-barrier defects with severe allergic manifestations. To aid in the development of a medicine for this target, an X-ray crystallographic system was developed to facilitate fragment-guided chemistry and knowledge-based drug-discovery approaches. Here, the development of a surrogate crystallographic system in place of KLK5, which proved to be challenging to crystallize, is described. The biochemical robustness of the crystallographic surrogate and the suitability of the system for the study of small nonpeptidic fragments and lead-like molecules are demonstrated.


Assuntos
Benzamidinas/química , Calicreínas/química , Inibidores de Proteases/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Benzamidinas/farmacologia , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Descoberta de Drogas , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Calicreínas/antagonistas & inibidores , Calicreínas/genética , Calicreínas/metabolismo , Cinética , Modelos Moleculares , Mutação , Síndrome de Netherton/tratamento farmacológico , Síndrome de Netherton/enzimologia , Inibidores de Proteases/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Eletricidade Estática , Especificidade por Substrato
9.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 392-396, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045569

RESUMO

Grx1, a cytosolic thiol-disulfide oxidoreductase, actively maintains cellular redox homeostasis using glutathione substrates (reduced, GSH, and oxidized, GSSG). Here, the crystallization of reduced Grx1 from the yeast Saccharomyces cerevisiae (yGrx1) in space group P212121 and its structure solution and refinement to 1.22 Šresolution are reported. To study the structure-function relationship of yeast Grx1, the crystal structure of reduced yGrx1 was compared with the existing structures of the oxidized and glutathionylated forms. These comparisons revealed structural differences in the conformations of residues neighbouring the Cys27-Cys30 active site which accompany alterations in the redox status of the protein.


Assuntos
Cisteína/química , Glutarredoxinas/química , Glutationa/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
10.
Mol Vis ; 25: 183-193, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30996587

RESUMO

Purpose: In Bornholm eye disease, a defect in the splicing of transcripts from a variant OPN1LW opsin gene leads to a depletion in spliced transcript levels and, consequently, a reduction in photopigment in photoreceptors expressing the variant gene. Methods: Myopic and age-matched control subjects were drawn from the Western Australian Pregnancy Cohort (Raine) Study and the Norfolk Island Eye Study groups. The OPN1LW opsin gene was amplified using long-range PCR methodology and was fully sequenced. Expression of variant opsins was evaluated using quantitative PCR (qPCR). RNA secondary structure changes arising from identified variants were predicted by modeling. Results: Forty-two nucleotide sites were found to vary across the 111 subjects studied. Of these, 15 had not been previously reported, with three present only in myopic individuals. Expression of these variants in transfected human embryonic kidney (HEK293T) cells demonstrated that splicing efficiencies were not affected. However, gene transcripts from two of the three variants were significantly depleted. RNA secondary structure modeling predicted that these single nucleotide changes could affect RNA stability. Conclusions: None of the variants identified in myopic individuals appeared to alter the efficiency of transcript splicing. However, two resulted in a significant reduction in the number of spliced and unspliced transcripts, indicating an overall reduction in steady-state transcript stability. Such a change would be expected to result in a reduced amount of photopigment, and this may be a contributing factor in the development of myopia.


Assuntos
Miopia/genética , Processamento de RNA , Estabilidade de RNA , RNA Mensageiro/genética , Opsinas de Bastonetes/genética , Adulto , Austrália , Estudos de Casos e Controles , Clonagem Molecular , Expressão Gênica , Variação Genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Ilhas , Masculino , Miopia/diagnóstico , Miopia/fisiopatologia , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Opsinas de Bastonetes/deficiência , Análise de Sequência de DNA
11.
Mol Biotechnol ; 61(6): 421-426, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30937688

RESUMO

The B-box proteins (BBXs) are zinc finger proteins containing one or two B-box domain(s) and involved in regulation of development processes as transcription factors in plants. Here, seven BBX genes in Malus domestica genome (MdBBXs) were identified and found to be up-regulated under abiotic stresses, with 2-12 folds in roots. All recombinant MdBBXs expressed in Escherichia coli (E. coli) enhanced the cell's tolerance to salt and osmotic stresses, respectively. Deficiency of B-box domain of MdBBX10 led to the loss of anti-stress functions. Five conservative cysteines in B-box domain played crucial roles in stress resistance, which are involved in two of metal iron binding sites of zinc finger motifs in BBXs. All the above results suggested MdBBXs confer stress tolerance to E. coli cell against abiotic stresses.


Assuntos
Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Malus/genética , Proteínas de Plantas/genética , Tolerância ao Sal/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Malus/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Polietilenoglicóis/farmacologia , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética
12.
Mol Biotechnol ; 61(6): 427-431, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30941576

RESUMO

Peroxisome proliferator-activated receptor gamma (PPARγ) is involved in the regulation of lipid and glucose homeostasis and inflammation. PPARγ expression level has been widely studied in multiple tissues; however, there are few reports of preceding attempts to produce full-length human PPARγ (hPPARγ) in cellular models, and generally, expression level is not known or measurable. We propose an alternative strategy to express recombinant hPPARγ1, using a transient transfection with an inducible Tet-On 3G system where target and reporter gene were cloned in the same open reading frame. We transiently co-transfected human embryonic kidney 293T (HEK293T) cells with pTRE-ZsGreen1-IRES2-hPPARγ1 and pCMV-TET3G for inducible expression of hPPARγ1. Relative expression of the transcript was evaluated by RT-qPCR 48 h after transfection, obtaining a high expression level of hPPARγ (530-fold change, p < 0.002) in co-transfected HEK293T cells in the presence of doxycycline (1 µg/mL); also a significantly increased production of the reporter protein ZsGreen1 (3.6-fold change, p < 0.05) was determined by fluorescence analysis. These data indicated that HEK293T cells were successfully co-transfected and it could be an alternative model for hPPARγ expression in vitro. Additionally, this model will help to validate the quantification of inducible hPPARγ expression in vivo models for future research.


Assuntos
Clonagem Molecular/métodos , Vetores Genéticos/metabolismo , PPAR gama/genética , Proteínas Recombinantes de Fusão/genética , Doxiciclina/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Vetores Genéticos/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Fases de Leitura Aberta , PPAR gama/biossíntese , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transfecção
13.
Mol Biotechnol ; 61(6): 400-409, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30945164

RESUMO

Transgenic chickens are of great interest for the production of recombinant proteins in their eggs. However, the use of constitutive strong promoters or the tissue-specific ovalbumin promoter for the generation of the transgenic chickens have different drawbacks that have to be overcome in order to make chicken bioreactor an efficient production system. This prompted us to investigate the use of an alternative tissue-specific promoter, the vitellogenin promoter, which could overcome the difficulties currently found in the generation of chicken bioreactors. In the present work we establish and characterize a DNA construct consisting of a fragment of the 5´-flanking region of the chicken vitellogenin II gene cloned in a reporter vector. This construct is capable of showing the ability of the promoter to drive expression of a reporting gene in a tissue-specific manner and in a way that closely resembles physiologic regulation of vitellogenin, making it an ideal candidate to be used in the future for generation of avian bioreactors. Besides, we validate an in vitro culture system to test the performance of the DNA construct under study that could be used as a practical tool before generating any transgenic chicken. These results are important since they provide the proof of concept for the use of the vitellogenin promoter for future genetic modification of chickens bioreactors with improved characteristics in terms of quality of the recombinant protein produced.


Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Vetores Genéticos/química , Proteínas Recombinantes de Fusão/genética , Vitelogeninas/genética , Região 5'-Flanqueadora , Animais , Animais Geneticamente Modificados , Proteínas Aviárias/metabolismo , Reatores Biológicos , Embrião de Galinha , Galinhas/metabolismo , Clonagem Molecular , Estradiol/farmacologia , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Luciferases/genética , Luciferases/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Receptores Estrogênicos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodos , Vitelogeninas/metabolismo , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
14.
Bioengineered ; 10(1): 108-120, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31017543

RESUMO

The granulocyte-macrophage colony-stimulating factor (GM-CSF) can be used to induce a powerful immune response. Based on the specific binding of biotin and streptavidin, SA-hGM-CSF was anchored on the surface of biotinylated tumor cells, which could enhance the anti-tumor effect of tumor cell vaccines in our previous reports, suggesting it would have potential clinical value. Preparation of the biologically active proteins in large-scale production is the basis of clinical application, however, only a small amount of biologically active protein was obtained according to previous studies. In this study, we researched the effects of various factors on the purification and simultaneous renaturation of SA-hGM-CSF fusion protein by single factor experiment and orthogonal experiment. Here, we developed a viable pilot-scale trial in the fermentation, purification, refolding and freeze-drying of SA-hGM-CSF proteins in order to efficiently obtain more biologically active proteins with high purity, which will lay the foundation for industrial production.


Assuntos
Biotina/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Proteínas Recombinantes de Fusão/genética , Estreptavidina/metabolismo , Sequência de Aminoácidos , Animais , Biotina/genética , Biotinilação , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Análise Fatorial , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Camundongos , Células PC-3 , Projetos Piloto , Desnaturação Proteica , Redobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Estreptavidina/genética
15.
Nat Commun ; 10(1): 1937, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028261

RESUMO

The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development.


Assuntos
DNA Nucleotidiltransferases/genética , DNA/genética , Edição de Genes/métodos , Genoma Humano , Integrases/genética , Sequência de Bases , Clonagem Molecular , DNA/metabolismo , DNA Nucleotidiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Integrases/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética
16.
Molecules ; 24(8)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013863

RESUMO

When studying polyethylenimine derivatives as nonviral vectors for gene delivery, among the important issues to be addressed are high toxicity, low transfection efficiency, and nucleic acid polyplex condensation. The molecular weight of polyethylenimine, PEGylation, biocompatibility and, also, supramolecular structure of potential carrier can all influence the nucleic acid condensation behavior, polyplex size, and transfection efficiency. The main challenge in building an efficient carrier is to find a correlation between the constituent components, as well as the synergy between them, to transport and to release, in a specific manner, different molecules of interest. In the present study, we investigated the synergy between components in dynamic combinatorial frameworks formed by connecting PEGylated squalene, poly-(ethyleneglycol)-bis(3-aminopropyl) and low molecular weight polyethylenimine components to 1,3,5-benzenetrialdehyde, via reversible imine bond, applying a dynamic combinatorial chemistry approach. We report comparative structural and morphological data, DNA binding affinity, toxicity and transfection efficiency concerning the ratio of polyethylenimine and presence or absence of poly-(ethyleneglycol)-bis(3-aminopropyl) in composition of dynamic combinatorial frameworks. In vitro biological assessments have revealed the fact that nonviral vectors containing poly-(ethyleneglycol)-bis(3-aminopropyl) and the lowest amount of polyethylenimine have significant transfection efficiency at N/P 50 ratio and display insignificant cytotoxicity on the HeLa cell line.


Assuntos
Vetores Genéticos , Polietilenoglicóis , Polietilenoimina , Transfecção/métodos , Sobrevivência Celular/efeitos dos fármacos , Vetores Genéticos/química , Vetores Genéticos/farmacologia , Células HeLa , Humanos , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Polietilenoimina/química , Polietilenoimina/farmacologia
17.
Mol Biotechnol ; 61(6): 410-420, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963479

RESUMO

Current developments in meta-data analysis and predictive computational models offer alternative routes for the identification of antibodies. In silico-based technologies and NGS data analysis from Ab phage-display selections offer expanded selections of Ab candidates. Accordingly, the identified de novo Abs with predicted selectivity for a target antigen must undergo rapid gene synthesis for downstream Ab characterizations. Here we describe a high-throughput strategy for the generation of synthetic Ab clones for expression as Fab proteins in Escherichia coli. Our approach utilizes simultaneous single-stranded site-directed mutagenesis of diversified Ab regions of a phagemid template with engineered complementary determining regions that contain multiple stop codon and restriction enzyme sites. Subsequently, we perform rapid screening of Ab DNA clones for correct gene assemblies by high-throughput Ab-phage protein expression screens. Identified sequences are corroborated by Sanger DNA sequencing analysis. In summary, our work describes a rapid and cost-effective platform for the high-throughput synthesis of synthetic Ab genes as Fab proteins for implementation into downstream protein validation pipelines.


Assuntos
Técnicas de Visualização da Superfície Celular , Clonagem Molecular/métodos , Vetores Genéticos/química , Ensaios de Triagem em Larga Escala , Anticorpos de Cadeia Única/genética , Códon de Terminação , Enzimas de Restrição do DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Anticorpos de Cadeia Única/biossíntese
18.
Mol Biotechnol ; 61(6): 432-441, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963480

RESUMO

D-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using D-psicose epimerase and L-rhamnulose kinase, a method enabling high conversion of D-allulose from D-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% D-fructose was achieved at the concentrations of 2 mM ATP, 5 mM polyphosphate, 20 mM D-fructose, and 20 mM Mg2+ when incubated at 50 °C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of D-fructose into D-allulose at reduced ATP cost.


Assuntos
Trifosfato de Adenosina/metabolismo , Carboidratos Epimerases/metabolismo , Frutose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Biotransformação , Carboidratos Epimerases/genética , Cátions Bivalentes , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Frutose/biossíntese , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Magnésio/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polifosfatos/metabolismo , Proteínas Recombinantes de Fusão/genética , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismo
19.
Mol Biotechnol ; 61(6): 451-460, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997666

RESUMO

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Nitrosomonas europaea/genética , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nitrosomonas europaea/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Periplasma/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo
20.
Mol Biotechnol ; 61(6): 461-468, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997667

RESUMO

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.


Assuntos
Proteínas de Bactérias/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , NADH NADPH Oxirredutases/genética , Fosfitos/metabolismo , Fósforo/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Proteínas de Bactérias/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Engenharia Genética/métodos , Marcadores Genéticos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fosfitos/farmacologia , Pseudomonas stutzeri/química , Pseudomonas stutzeri/genética , Seleção Genética , Transformação Genética
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