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1.
PLoS One ; 18(9): e0291131, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37729215

RESUMO

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery.


Assuntos
Antígenos de Grupos Sanguíneos , COVID-19 , Humanos , SARS-CoV-2 , Vacinas contra COVID-19 , Anticorpos Antivirais , Anticorpos Neutralizantes , Epitopos
2.
JHEP Rep ; 5(4): 100651, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36866391

RESUMO

Background & Aims: Oxidative stress is recognized as a major driver of non-alcoholic steatohepatitis (NASH) progression. The transcription factor NRF2 and its negative regulator KEAP1 are master regulators of redox, metabolic and protein homeostasis, as well as detoxification, and thus appear to be attractive targets for the treatment of NASH. Methods: Molecular modeling and X-ray crystallography were used to design S217879 - a small molecule that could disrupt the KEAP1-NRF2 interaction. S217879 was highly characterized using various molecular and cellular assays. It was then evaluated in two different NASH-relevant preclinical models, namely the methionine and choline-deficient diet (MCDD) and diet-induced obesity NASH (DIO NASH) models. Results: Molecular and cell-based assays confirmed that S217879 is a highly potent and selective NRF2 activator with marked anti-inflammatory properties, as shown in primary human peripheral blood mononuclear cells. In MCDD mice, S217879 treatment for 2 weeks led to a dose-dependent reduction in NAFLD activity score while significantly increasing liver Nqo1 mRNA levels, a specific NRF2 target engagement biomarker. In DIO NASH mice, S217879 treatment resulted in a significant improvement of established liver injury, with a clear reduction in both NAS and liver fibrosis. αSMA and Col1A1 staining, as well as quantification of liver hydroxyproline levels, confirmed the reduction in liver fibrosis in response to S217879. RNA-sequencing analyses revealed major alterations in the liver transcriptome in response to S217879, with activation of NRF2-dependent gene transcription and marked inhibition of key signaling pathways that drive disease progression. Conclusions: These results highlight the potential of selective disruption of the NRF2-KEAP1 interaction for the treatment of NASH and liver fibrosis. Impact and implications: We report the discovery of S217879 - a potent and selective NRF2 activator with good pharmacokinetic properties. By disrupting the KEAP1-NRF2 interaction, S217879 triggers the upregulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved in NASH disease progression, leading ultimately to the reduction of both NASH and liver fibrosis progression in mice.

3.
Bioorg Med Chem Lett ; 75: 128950, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36030002

RESUMO

We describe the synthesis of a series of 3-t-butyl 5-aminopyrazole p-substituted arylamides as inhibitors of serine-threonine25 (STK25), an enzyme implicated in the progression of non-alcoholic fatty liver disease (NAFLD). Appending a p-N-pyrrolidinosulphonamide group to the arylamide group led to a 'first-in kind' inhibitor with IC50 = 228 nM. A co-crystal structure with STK 25 revealed productive interactions which were also reproduced using molecular docking. A new series of triazolo dihydro oxazine carboxamides of 3-t-butyl 5-aminopyrazole was not active against STK25.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Simulação de Acoplamento Molecular , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Oxazinas , Proteínas Serina-Treonina Quinases , Serina , Treonina , Raios X
4.
ACS Med Chem Lett ; 13(6): 949-954, 2022 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-35707140

RESUMO

On the basis of the knowledge that the proline-rich hot spot PPPRPP region of P(151)PSNPPPRPP(160), an oligopeptide derived from the cytosolic portion of p22phox (p22), binds to the single functional bis-SH3 domain of the regulatory protein p47phox (p47), we designed a mimetic of the tripeptide PPP based on NMR and X-ray crystallographic data for the p22(151-161) peptide PPSNPPPRPPA with a peptide construct. Incorporation of the synthetic pseudo-triproline mimetic Pro-Pro-Cyp in a molecule derived from molecular modeling studies led to only a 7-fold diminution in activity in a surface plasmon resonance assay relative to the same molecule containing the natural Pro-Pro-Pro tripeptide. The alternative sequence corresponding to a Pro-Cyp-Pro insertion was inactive. This is a first example of the use of a triproline mimetic to interfere with the formation of the p47-p22 complex, which is critical for the activation of NOX, leading to the production of reactive oxygen species as superoxide anions.

5.
J Biol Chem ; 294(10): 3720-3734, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30598509

RESUMO

Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid-binding protein 1 (FABP1), GW7647 treatment increases FABP1's nuclear localization and potentiates GW7647-mediated PPARα activation; GW7647 is less effective in cells that do not express FABP1. To elucidate the underlying mechanism, here we substituted residues in FABP1 known to dictate lipid signaling by other intracellular lipid-binding proteins. Substitutions of Lys-20 and Lys-31 to Ala in the FABP1 helical cap affected neither its nuclear localization nor PPARα activation. In contrast, Ala substitution of Lys-57, Glu-77, and Lys-96, located in the loops adjacent to the ligand-binding portal region, abolished both FABP1 nuclear localization and GW7647-induced PPARα activation but had little effect on GW7647-FABP1 binding affinity. Using solution NMR spectroscopy, we determined the WT FABP1 structure and analyzed the dynamics in the apo and GW7647-bound structures of both the WT and the K57A/E77A/K96A triple mutant. We found that GW7647 binding causes little change in the FABP1 backbone, but solvent exposes several residues in the loops around the portal region, including Lys-57, Glu-77, and Lys-96. These residues also become more solvent-exposed upon binding of FABP1 with the endogenous PPARα agonist oleic acid. Together with previous observations, our findings suggest that GW7647 binding stabilizes a FABP1 conformation that promotes its interaction with PPARα. We conclude that full PPARα agonist activity of GW7647 requires FABP1-dependent transport and nuclear localization processes.


Assuntos
Butiratos/farmacologia , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , PPAR alfa/agonistas , Compostos de Fenilureia/farmacologia , Butiratos/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Ligantes , Modelos Moleculares , Mutação , Compostos de Fenilureia/metabolismo , Conformação Proteica/efeitos dos fármacos
6.
J Biol Chem ; 293(23): 9064-9077, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29695506

RESUMO

The GTPase RhoA is a major player in many different regulatory pathways. RhoA catalyzes GTP hydrolysis, and its catalysis is accelerated when RhoA forms heterodimers with proteins of the guanine nucleotide exchange factor (GEF) family. Neuroepithelial cell transforming gene 1 (Net1) is a RhoA-interacting GEF implicated in cancer, but the structural features supporting the RhoA/Net1 interaction are unknown. Taking advantage of a simple production and purification process, here we solved the structure of a RhoA/Net1 heterodimer with X-ray crystallography at 2-Å resolution. Using a panel of several techniques, including molecular dynamics simulations, we characterized the RhoA/Net1 interface. Moreover, deploying an extremely simple peptide-based scanning approach, we found that short peptides (penta- to nonapeptides) derived from the protein/protein interaction region of RhoA could disrupt the RhoA/Net1 interaction and thereby diminish the rate of nucleotide exchange. The most inhibitory peptide, EVKHF, spanning residues 102-106 in the RhoA sequence, displayed an IC50 of ∼100 µm without further modifications. The peptides identified here could be useful in further investigations of the RhoA/Net1 interaction region. We propose that our structural and functional insights might inform chemical approaches for transforming the pentapeptide into an optimized pseudopeptide that antagonizes Net1-mediated RhoA activation with therapeutic anticancer potential.


Assuntos
Proteínas Oncogênicas/química , Proteína rhoA de Ligação ao GTP/química , Sequência de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacologia , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Proteínas Oncogênicas/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Alinhamento de Sequência , Proteína rhoA de Ligação ao GTP/metabolismo
7.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 10): 574-578, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28994406

RESUMO

A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.


Assuntos
Microfluídica/métodos , Difração de Raios X/métodos , Cristalização/instrumentação , Cristalização/métodos , Microfluídica/instrumentação , Difração de Raios X/instrumentação
8.
Cereb Cortex ; 27(12): 5635-5651, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28968740

RESUMO

Planar cell polarity (PCP) signaling is well known to play a critical role during prenatal brain development; whether it plays specific roles at postnatal stages remains rather unknown. Here, we investigated the role of a key PCP-associated gene scrib in CA1 hippocampal structure and function at postnatal stages. We found that Scrib is required for learning and memory consolidation in the Morris water maze as well as synaptic maturation and NMDAR-dependent bidirectional plasticity. Furthermore, we unveiled a direct molecular interaction between Scrib and PP1/PP2A phosphatases whose levels were decreased in postsynaptic density of conditional knock-out mice. Remarkably, exposure to enriched environment (EE) preserved memory formation in CaMK-Scrib-/- mice by recovering synaptic plasticity and maturation. Thus, Scrib is required for synaptic function involved in memory formation and EE has beneficiary therapeutic effects. Our results demonstrate a distinct new role for a PCP-associated protein, beyond embryonic development, in cognitive functions during adulthood.


Assuntos
Disfunção Cognitiva/fisiopatologia , Disfunção Cognitiva/terapia , Meio Ambiente , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Plasticidade Neuronal/fisiologia , Animais , Células COS , Chlorocebus aethiops , Disfunção Cognitiva/patologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Abrigo para Animais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Deficiências da Aprendizagem/patologia , Deficiências da Aprendizagem/fisiopatologia , Deficiências da Aprendizagem/terapia , Masculino , Transtornos da Memória/patologia , Transtornos da Memória/fisiopatologia , Transtornos da Memória/terapia , Camundongos Knockout , Modelos Moleculares , Densidade Pós-Sináptica/metabolismo , Densidade Pós-Sináptica/ultraestrutura , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura
9.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 4): 174-183, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28368275

RESUMO

The invention of the electron microscope has greatly enhanced the view scientists have of small structural details. Since its implementation, this technology has undergone considerable evolution and the resolution that can be obtained for biological objects has been extended. In addition, the latest generation of cryo-electron microscopes equipped with direct electron detectors and software for the automated collection of images, in combination with the use of advanced image-analysis methods, has dramatically improved the performance of this technique in terms of resolution. While calculating a sub-10 Šresolution structure was an accomplishment less than a decade ago, it is now common to generate structures at sub-5 Šresolution and even better. It is becoming possible to relatively quickly obtain high-resolution structures of biological molecules, in particular large ones (>500 kDa) which, in some cases, have resisted more conventional methods such as X-ray crystallography or nuclear magnetic resonance (NMR). Such newly resolved structures may, for the first time, shed light on the precise mechanisms that are essential for cellular physiological processes. The ability to attain atomic resolution may support the development of new drugs that target these proteins, allowing medicinal chemists to understand the intimacy of the relationship between their molecules and targets. In addition, recent developments in cryo-electron microscopy combined with image analysis can provide unique information on the conformational variability of macromolecular complexes. Conformational flexibility of macromolecular complexes can be investigated using cryo-electron microscopy and multiconformation reconstruction methods. However, the biochemical quality of the sample remains the major bottleneck to routine cryo-electron microscopy-based determination of structures at very high resolution.


Assuntos
Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Drogas em Investigação/química , Substâncias Macromoleculares/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Microscopia Crioeletrônica/instrumentação , Cristalização , Cristalografia por Raios X/instrumentação , Descoberta de Drogas , Processamento de Imagem Assistida por Computador , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Conformação Molecular , Canal de Liberação de Cálcio do Receptor de Rianodina/ultraestrutura
10.
Protein Sci ; 25(12): 2225-2242, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27670942

RESUMO

Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.


Assuntos
Redobramento de Proteína , Proteínas de Ligação a Tacrolimo , Cristalografia por Raios X , Humanos , Domínios Proteicos , Relação Estrutura-Atividade , Proteínas de Ligação a Tacrolimo/síntese química , Proteínas de Ligação a Tacrolimo/química
11.
Med Sci (Paris) ; 32(8-9): 758-67, 2016.
Artigo em Francês | MEDLINE | ID: mdl-27615185

RESUMO

Recent technological advances have revolutionized the field of structural biologists. Specifically, dramatic progress related to the development of new electron microscopes and image capture (direct electron detection camera) and the provision of new image analysis software has led to a breakthrough in terms of resolution attained using cryo-electron transmission microscopy. It is thus possible to calculate relatively quickly high-resolution structures of biological molecules whom structural study still resists to more conventional methods such as X-ray diffraction or nuclear magnetic resonance (NMR). These structures thus obtained may also bring complementary structural information to those already described by other methods. Some of these new structures resolved through cryo-electron microscopy revealed for the first time the precise operation of essential mechanisms necessary for the good physiological process of a cell. The ability to solve these structures at atomic resolution detail is essential for the development of new drugs that target these proteins of therapeutic interest. Thanks to these advanced techniques that we summarize in this revew, biological and medical issues have now become accessible, whereas this approach was inconceivable only five yeras ago. ‡.


Assuntos
Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de Transmissão/métodos , Conformação Proteica , Proteínas/química , Animais , Cristalografia por Raios X , Humanos , Processamento de Imagem Assistida por Computador/métodos , Modelos Moleculares
12.
Cell Rep ; 9(2): 712-27, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25310985

RESUMO

The appropriate trafficking of glutamate receptors to synapses is crucial for basic synaptic function and synaptic plasticity. It is now accepted that NMDA receptors (NMDARs) internalize and are recycled at the plasma membrane but also exchange between synaptic and extrasynaptic pools; these NMDAR properties are also key to governing synaptic plasticity. Scribble1 is a large PDZ protein required for synaptogenesis and synaptic plasticity. Herein, we show that the level of Scribble1 is regulated in an activity-dependent manner and that Scribble1 controls the number of NMDARs at the plasma membrane. Notably, Scribble1 prevents GluN2A subunits from undergoing lysosomal trafficking and degradation by increasing their recycling to the plasma membrane following NMDAR activation. Finally, we show that a specific YxxR motif on Scribble1 controls these mechanisms through a direct interaction with AP2. Altogether, our findings define a molecular mechanism to control the levels of synaptic NMDARs via Scribble1 complex signaling.


Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Endossomos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Dados de Sequência Molecular , Neurônios/metabolismo , Ligação Proteica , Transporte Proteico , Proteólise , Ratos , Ratos Sprague-Dawley , Proteínas Supressoras de Tumor/química
13.
Acta Crystallogr D Biol Crystallogr ; 67(Pt 11): 929-35, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22101819

RESUMO

Glucokinase (GK) catalyses the formation of glucose 6-phosphate from glucose and ATP. A specific feature of GK amongst hexokinases is that it can cycle between active and inactive conformations as a function of glucose concentration, resulting in a unique positive kinetic cooperativity with glucose, which turns GK into a unique key sensor of glucose metabolism, notably in the pancreas. GK is a target of antidiabetic drugs aimed at the activation of GK activity, leading to insulin secretion. Here, the first structures of a GK-glucose complex without activator, of GK-glucose-AMP-PNP and of GK-glucose-AMP-PNP with a bound activator are reported. All these structures are extremely similar, thus demonstrating that binding of GK activators does not result in conformational changes of the active protein but in stabilization of the active form of GK.


Assuntos
Hiperinsulinismo Congênito/tratamento farmacológico , Glucoquinase/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Regulação Alostérica/efeitos dos fármacos , Hiperinsulinismo Congênito/metabolismo , Hiperinsulinismo Congênito/patologia , Cristalização , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Glucoquinase/química , Glucose/análogos & derivados , Glucose/química , Humanos , Hipoglicemiantes/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos
14.
J Biol Chem ; 285(39): 30126-38, 2010 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-20592031

RESUMO

We present an interdisciplinary approach that, by incorporating a range of experimental and computational techniques, allows the identification and characterization of functional/immunogenic domains. This approach has been applied to ArtJ, an arginine-binding protein whose orthologs in Chlamydiae trachomatis (CT ArtJ) and pneumoniae (CPn ArtJ) are shown to have different immunogenic properties despite a high sequence similarity (60% identity). We have solved the crystallographic structures of CT ArtJ and CPn ArtJ, which are found to display a type II transporter fold organized in two α-ß domains with the arginine-binding region at their interface. Although ArtJ is considered to belong to the periplasm, we found that both domains contain regions exposed on the bacterial surface. Moreover, we show that recombinant ArtJ binds to epithelial cells in vitro, suggesting a role for ArtJ in host-cell adhesion during Chlamydia infection. Experimental epitope mapping and computational analysis of physicochemical determinants of antibody recognition revealed that immunogenic epitopes reside mainly in the terminal (D1) domain of both CPn and CT ArtJ, whereas the surface properties of the respective binding-prone regions appear sufficiently different to assume divergent immunogenic behavior. Neutralization assays revealed that sera raised against CPn ArtJ D1 partially reduce both CPn and CT infectivity in vitro, suggesting that functional antibodies directed against this domain may potentially impair chlamydial infectivity. These findings suggest that the approach presented here, combining functional and structure-based analyses of evolutionary-related antigens can be a valuable tool for the identification of cross-species immunogenic epitopes for vaccine development.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/química , Proteínas de Bactérias/química , Vacinas Bacterianas/química , Chlamydia trachomatis/química , Chlamydophila pneumoniae/química , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/imunologia , Aderência Bacteriana/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Infecções por Chlamydia/prevenção & controle , Chlamydia trachomatis/genética , Chlamydia trachomatis/imunologia , Infecções por Chlamydophila/prevenção & controle , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/imunologia , Cristalografia por Raios X , Mapeamento de Epitopos/métodos , Estrutura Terciária de Proteína
15.
J Mol Biol ; 343(2): 407-16, 2004 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-15451669

RESUMO

Human BACE, also known as beta-secretase, shows promise as a potential therapeutic target for Alzheimer's disease. We determined the apo structure of BACE to 1.75 A, and a structure of a hydroxyethylamine inhibitor complex derived by soaking. These show significant active-site movements compared to previously described BACE structures. Additionally, the structures reveal two pockets that could be targeted by structure-based drug design.


Assuntos
Aminas/química , Endopeptidases/química , Inibidores Enzimáticos/química , Estrutura Terciária de Proteína , Aminas/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Endopeptidases/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Ligação de Hidrogênio , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Temperatura
16.
Protein Expr Purif ; 37(1): 203-6, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15294299

RESUMO

Two mutant strains of Escherichia coli BL21(DE3), called C41(DE3) and C43(DE3) and originally described by Miroux and Walker, are frequently used to overcome the toxicity associated with overexpressing recombinant proteins using the bacteriophage T7 RNA polymerase expression system. Even when the toxicity of the plasmids is so high that it prevents transformation in the strain BL21(DE3), the toxic proteins can often be expressed successfully in C41(DE3) and/or C43(DE3). In this work, using a range of plasmids coding for several types of proteins, we investigated in BL21(DE3), C41(DE3), and C43(DE3) their ability to undergo transformation and to express. While transformation was always possible in C41(DE3) and C43(DE3), we could not obtain transformants in BL21(DE3) for 62% of the expression vectors tested. Moreover, after induction, the expression of heterologous proteins in both mutant strains is generally better than in BL21(DE3). In this study, we also enhanced the stability of plasmids in culture during the expression of proteins by adding the par locus from the plasmid pSC101 to the vector backbone. The stability of a subset of the plasmids (measured 3 h after induction) was determined in C41(DE3) and C43(DE3) and varies from 62 to 92% for C43(DE3) and from 10 to 90% for C41(DE3). This study demonstrates the usefulness of these strains C41(DE3) and C43(DE3) in solving the problem of plasmid instability during the expression of toxic recombinant proteins.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos/toxicidade , Proteínas Recombinantes/toxicidade , Transformação Genética , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
Biophys Chem ; 100(1-3): 469-79, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12646384

RESUMO

Some non-detergent sulfobetaines had been shown to prevent aggregation and improve the yield of active proteins when added to the buffer during in vitro protein renaturation. With the aim of designing more efficient folding helpers, a series of non-detergent sulfobetaines have been synthesized and their efficiency in improving the renaturation of a variety of proteins (E. coli tryptophan synthase and beta-D-galactosidase, hen lysozyme, bovine serum albumin, a monoclonal antibody) have been investigated. Attempts to correlate the structure of each sulfobetaines with its effect on folding revealed some molecular features that appear important in helping renaturation. This enabled us to design and synthesize new non-detergent sulfobetaines that act as potent folding helpers.


Assuntos
Betaína/análogos & derivados , Dobramento de Proteína , Animais , Anticorpos Bloqueadores/química , Anticorpos Monoclonais/química , Betaína/química , Bovinos , Fenômenos Químicos , Físico-Química , Galinhas , Desenho de Fármacos , Camundongos , Muramidase/química , Renaturação Proteica , Proteínas/química , Soroalbumina Bovina/química , Triptofano Sintase/antagonistas & inibidores , Triptofano Sintase/química
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