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1.
PLoS One ; 16(6): e0252507, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34061896

RESUMO

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Assuntos
Teste para COVID-19/normas , COVID-19/diagnóstico , COVID-19/epidemiologia , Testes Diagnósticos de Rotina/normas , Indicadores e Reagentes/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , SARS-CoV-2/genética , COVID-19/virologia , Teste para COVID-19/métodos , Camarões/epidemiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Gana/epidemiologia , Humanos , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Indicadores e Reagentes/provisão & distribuição , Técnicas de Diagnóstico Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Biologia Sintética/métodos , Transformação Bacteriana , Reino Unido/epidemiologia
2.
Appl Microbiol Biotechnol ; 105(11): 4635-4648, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34059939

RESUMO

Currently, the lack of reliable strategies for the diagnosis and treatment of cancer makes the identification and characterization of new therapeutic targets a pressing matter. Several studies have proposed the Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) as a promising therapeutic target for prostate cancer. Although structural and functional studies may provide deeper insights on the role of STEAP1 in cancer, such techniques require high amounts of purified protein through biotechnological processes. Based on the results presented, this work proposes the application, for the first time, of a fed-batch profile to improve STEAP1 biosynthesis in mini-bioreactor Komagataella pastoris X-33 Mut+ methanol-induced cultures, by evaluating three glycerol feeding profiles-constant, exponential, and gradient-during the pre-induction phase. Interestingly, different glycerol feeding profiles produced differently processed STEAP1. This platform was optimized using a combination of chemical chaperones for ensuring the structural stabilization and appropriate processing of the target protein. The supplementation of culture medium with 6 % (v/v) DMSO and 1 M proline onto a gradient glycerol/constant methanol feeding promoted increased biosynthesis levels of STEAP1 and minimized aggregation events. Deglycosylation assays with peptide N-glycosidase F showed that glycerol constant feed is associated with an N-glycosylated pattern of STEAP1. The biological activity of recombinant STEAP1 was also validated, once the protein enhanced the proliferation of LNCaP and PC3 cancer cells, in comparison with non-tumoral cell cultures. This methodology could be a crucial starting point for large-scale production of active and stable conformation of recombinant human STEAP1. Thus, it could open up new strategies to unveil the structural rearrangement of STEAP1 and to better understand the biological role of the protein in cancer onset and progression.


Assuntos
Antígenos de Neoplasias/biossíntese , Glicerol , Metanol , Oxirredutases/biossíntese , Proteínas Recombinantes/biossíntese , Humanos , Pichia , Regiões Promotoras Genéticas , Saccharomycetales
3.
Appl Microbiol Biotechnol ; 105(11): 4515-4534, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34059941

RESUMO

In the past decades, the production of biopharmaceuticals has gained high interest due to its great sensitivity, specificity, and lower risk of negative effects to patients. Biopharmaceuticals are mostly therapeutic recombinant proteins produced through biotechnological processes. In this context, L-asparaginase (L-asparagine amidohydrolase, L-ASNase (E.C. 3.5.1.1)) is a therapeutic enzyme that has been abundantly studied by researchers due to its antineoplastic properties. As a biopharmaceutical, L-ASNase has been used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), and other lymphoid malignancies, in combination with other drugs. Besides its application as a biopharmaceutical, this enzyme is widely used in food processing industries as an acrylamide mitigation agent and as a biosensor for the detection of L-asparagine in physiological fluids at nano-levels. The great demand for L-ASNase is supplied by recombinant enzymes from Escherichia coli and Erwinia chrysanthemi. However, production processes are associated to low yields and proteins associated to immunogenicity problems, which leads to the search for a better enzyme source. Considering the L-ASNase pharmacological and food importance, this review provides an overview of the current biotechnological developments in L-ASNase production and biochemical characterization aiming to improve the knowledge about its production. KEY POINTS: • Microbial enzyme applications as biopharmaceutical and in food industry • Biosynthesis process: from the microorganism to bioreactor technology • Enzyme activity and kinetic properties: crucial for the final application.


Assuntos
Antineoplásicos/metabolismo , Asparaginase/biossíntese , Asparagina , Biotecnologia , Dickeya chrysanthemi , Escherichia coli , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Recombinantes/biossíntese
4.
Braz J Biol ; 82: e244735, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34076169

RESUMO

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G - 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.


Assuntos
Antineoplásicos/farmacologia , Asparaginase , Pyrococcus abyssi , Asparaginase/biossíntese , Asparaginase/farmacologia , Células CACO-2 , Estabilidade Enzimática , Escherichia coli/genética , Humanos , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Pyrococcus abyssi/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Especificidade por Substrato
5.
Sci Rep ; 11(1): 10475, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006961

RESUMO

Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.


Assuntos
SARS-CoV-2/metabolismo , Anticorpos de Cadeia Única/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Reações Antígeno-Anticorpo , COVID-19/tratamento farmacológico , COVID-19/virologia , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Testes de Neutralização , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/uso terapêutico , Ressonância de Plasmônio de Superfície , Proteína Supressora de Tumor p53/química
6.
J Med Chem ; 64(9): 5838-5849, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33876629

RESUMO

Sirtuins are signaling hubs orchestrating the cellular response to various stressors with roles in all major civilization diseases. Sirtuins remove acyl groups from lysine residues of proteins, thereby controlling their activity, turnover, and localization. The seven human sirtuins, SirT1-7, are closely related in structure, hindering the development of specific inhibitors. Screening 170,000 compounds, we identify and optimize SirT1-specific benzoxazine inhibitors, Sosbo, which rival the efficiency and surpass the selectivity of selisistat (EX527). The compounds inhibit the deacetylation of p53 in cultured cells, demonstrating their ability to permeate biological membranes. Kinetic analysis of inhibition and docking studies reveal that the inhibitors bind to a complex of SirT1 and nicotinamide adenine dinucleotide, similar to selisistat. These new SirT1 inhibitors are valuable alternatives to selisistat in biochemical and cell biological studies. Their greater selectivity may allow the development of better targeted drugs to combat SirT1 activity in diseases such as cancer, Huntington's chorea, or anorexia.


Assuntos
Benzoxazinas/química , Sirtuína 1/antagonistas & inibidores , Acetilação/efeitos dos fármacos , Amidas/química , Benzoxazinas/metabolismo , Benzoxazinas/farmacologia , Sítios de Ligação , Carbazóis/química , Carbazóis/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Cinética , Simulação de Acoplamento Molecular , NAD/química , NAD/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Sirtuína 1/genética , Sirtuína 1/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/metabolismo
7.
Food Chem ; 355: 129462, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-33848938

RESUMO

Development of a high-performance chitinase for efficient biotransformation of insoluble chitinous substrate would be highly valuable in industry. In this study, the chitin-binding domains (ChBDs) of chitinase SaChiA4 were successfully modified to improve the enzymatic activity. The engineered substitution variant R-SaChiA4, which had the exogenous ChBD of chitinase ChiA1 from Bacillus circulans WL-12 (ChBDChiA1) substituted for its original ChBDChiA4, increased its activity by nearly 54% (28.0 U/mg) towards chitin powder, and by 49% towards colloidal chitin, compared with the wild-type. The substrate-binding assay demonstrated that the ChBD could enhance the capacity of enzymatic hydrolysis by promoting substrate affinity, and molecular dynamics simulations indicated that this could be due to hydrophobic interactions in different substrate binding modes. This work advances the understanding of the role of the ChBD, and provides a step towards the achievement of industrial-scale hydrolysis and utilization of insoluble chitin.


Assuntos
Proteínas de Bactérias/metabolismo , Quitina/metabolismo , Quitinases/metabolismo , Engenharia de Proteínas , Sequência de Aminoácidos , Bacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Quitinases/química , Quitinases/genética , Hidrólise , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato
8.
Int J Mol Sci ; 22(7)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916093

RESUMO

Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (MutS) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/Lmedium/h was achieved, which was 102.5-fold higher compared to flask cultivations.


Assuntos
Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/genética , Saccharomycetales/metabolismo , Proteínas Recombinantes/biossíntese
9.
J Med Chem ; 64(8): 4462-4477, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33793216

RESUMO

A ligand-based approach involving systematic modifications of a trisubstituted pyrazoline scaffold derived from the COX2 inhibitor, celecoxib, was used to develop novel PDE5 inhibitors. Novel pyrazolines were identified with potent PDE5 inhibitory activity lacking COX2 inhibitory activity. Compound d12 was the most potent with an IC50 of 1 nM, which was three times more potent than sildenafil and more selective with a selectivity index of >10,000-fold against all other PDE isozymes. Sildenafil inhibited the full-length and catalytic fragment of PDE5, while compound d12 only inhibited the full-length enzyme, suggesting a mechanism of enzyme inhibition distinct from sildenafil. The PDE5 inhibitory activity of compound d12 was confirmed in cells using a cGMP biosensor assay. Oral administration of compound d12 achieved plasma levels >1000-fold higher than IC50 values and showed no discernable toxicity after repeated dosing. These results reveal a novel strategy to inhibit PDE5 with unprecedented potency and isozyme selectivity.


Assuntos
Celecoxib/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Inibidores da Fosfodiesterase 5/química , Pirazóis/química , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Celecoxib/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Desenho de Fármacos , Feminino , Meia-Vida , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Inibidores da Fosfodiesterase 5/metabolismo , Ligação Proteica , Pirazóis/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Estereoisomerismo , Relação Estrutura-Atividade
10.
Food Chem ; 355: 129586, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-33773458

RESUMO

In order to invent a porcine gelatine detection device using microbial resources, bacterial enzymes with a preference towards porcine gelatine and their candidate genes were evaluated. Five (n = 5) bacterial strains isolated from hot spring water and wet clay, Malaysia were screened for their gelatinase activity. The gelatinase enzyme was extracted and purified using ammonium sulphate precipitation prior to performing gelatinase assay on porcine, bovine and fish gelatine medium substrates. The G2 strain or Enterobacter aerogenes (Strain EA1) was selected for whole genome sequenced after showing a consistent trend of preference towards porcine gelatine. The gelatinase candidate gene gelEA1_9 was cloned and expressed. Based on one-way analysis of variance (ANOVA) with POST-HOC Duncan test (α = 0.05), the final product of gelEA1_9 was identified as a novel gelatinase. This gelatinase presented no significant difference in activity towards porcine gelatine. Hence, the present study demonstrated an enzyme-substrate interaction for porcine gelatine identification.


Assuntos
Proteínas de Bactérias/metabolismo , Gelatina/metabolismo , Gelatinases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bovinos , Enterobacter aerogenes/enzimologia , Enterobacter aerogenes/genética , Peixes/metabolismo , Gelatinases/química , Gelatinases/genética , Expressão Gênica , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Especificidade por Substrato , Suínos
11.
Eur J Med Chem ; 216: 113296, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33677352

RESUMO

Acid (AC), neutral (NC) and alkaline ceramidase 3 (ACER3) are the most ubiquitous ceramidases and their therapeutic interest as targets in cancer diseases has been well sustained. This supports the importance of discovering potent and specific inhibitors for further use in combination therapies. Although several ceramidase inhibitors have been reported, most of them target AC and a few focus on NC. In contrast, well characterized ACER3 inhibitors are lacking. Here we report on the synthesis and screening of two series of 1-deoxy(dihydro)ceramide analogs on the three enzymes. Activity was determined using fluorogenic substrates in recombinant human NC (rhNC) and both lysates and intact cells enriched in each enzyme. None of the molecules elicited a remarkable AC inhibitory activity in either experimental setup, while using rhNC, several compounds of both series were active as non-competitive inhibitors with Ki values between 1 and 5 µM. However, a dramatic loss of potency occurred in NC-enriched cell lysates and no activity was elicited in intact cells. Interestingly, several compounds of Series 2 inhibited ACER3 dose-dependently in both cell lysates and intact cells with IC50's around 20 µM. In agreement with their activity in live cells, they provoked a significant increase in the amounts of ceramides. Overall, this study identifies highly selective ACER3 activity blockers in intact cells, opening the door to further medicinal chemistry efforts aimed at developing more potent and specific compounds.


Assuntos
Ceramidase Alcalina/antagonistas & inibidores , Ceramidas/química , Ceramidase Alcalina/genética , Ceramidase Alcalina/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/metabolismo , Ceramidas/farmacologia , Cromatografia Líquida de Alta Pressão , Avaliação Pré-Clínica de Medicamentos , Humanos , Cinética , Espectrometria de Massas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Esfingolipídeos/análise , Especificidade por Substrato
12.
Carbohydr Polym ; 260: 117814, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33712158

RESUMO

Lytic polysaccharide monooxygenases (LPMOs), monocopper enzymes that oxidatively cleave recalcitrant polysaccharides, have important biotechnological applications. Thermothelomyces thermophilus is a rich source of biomass-active enzymes, including many members from auxiliary activities family 9 LPMOs. Here, we report biochemical and structural characterization of recombinant TtLPMO9H which oxidizes cellulose at the C1 and C4 positions and shows enhanced activity in light-driven catalysis assays. TtLPMO9H also shows activity against xyloglucan. The addition of TtLPMO9H to endoglucanases from four different glucoside hydrolase families (GH5, GH12, GH45 and GH7) revealed that the product formation was remarkably increased when TtLPMO9H was combined with GH7 endoglucanase. Finally, we determind the first low resolution small-angle X-ray scattering model of the two-domain TtLPMO9H in solution that shows relative positions of its two functional domains and a conformation of the linker peptide, which can be relevant for the catalytic oxidation of cellulose and xyloglucan.


Assuntos
Celulases/metabolismo , Celulose/metabolismo , Ativação Enzimática/efeitos da radiação , Proteínas Fúngicas/metabolismo , Luz , Oxigenases de Função Mista/metabolismo , Sordariales/enzimologia , Biomassa , Catálise , Celulose/química , Proteínas Fúngicas/química , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Glucanos/química , Glucanos/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/classificação , Oxigenases de Função Mista/genética , Oxirredução , Filogenia , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espalhamento a Baixo Ângulo , Estereoisomerismo , Especificidade por Substrato , Difração de Raios X , Xilanos/química , Xilanos/metabolismo
13.
Chem Pharm Bull (Tokyo) ; 69(2): 178-184, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33518600

RESUMO

C1q/tumor necrosis factor (TNF)-related protein 12 (CTRP12) plays a crucial part in cardiovascular diseases especially the coronary artery disease. Nonetheless, it is unrevealed that whether the CTRP12 participates in the progress of cardiac fibrosis. In this study, we investigated whether CTRP12 regulates pathological myocardial fibrosis. We isolated neonatal rat cardiac fibroblasts were cultured with recombination CTRP12 followed by stimulating with Isoproterenol (ISO, 100 µM) for 24 h. Then the adenovirus were used to achieve the CTRP12-overexpressed fibroblasts. In vivo, the C57/B6 mice were subjected to recombinant human CTRP12 (0.2 µg/g/d) for 2 weeks after injected with Isoproterenol (ISO, 10 mg/kg/d for 3 d then 5 mg/kg/d for 11 d, subcutaneously (s.c.), 2 weeks) and mice were also subjected to adenovirus with P38 overexpressing system to explore the mechanism. As a result, CTRP12 significantly inhibit the transformation of cardiac fibroblasts to myofibroblasts and the transcription of cardiac fibrosis-related proteins induced by ISO in vitro. The administration of CTRP12 can effectively reduce the cardiac fibrosis and enhance the cardiac function in mice hearts. The treatment with CTRP12 did not change the expression level of phosphorylated (p)-smad2, smad4, p-extracellular regulated protein kinases 1/2 and c-Jun N-terminal kinase 1/2, but it suppressed the activation of p38. Cardiac overexpression of p38 could abolish this kind of cardioprotective effects by CTRP12. In summary, the CTRP12 protect against the ISO induced cardiac fibrosis via suppressing the p38 signal pathway.


Assuntos
Adipocinas/farmacologia , Isoproterenol/toxicidade , Transdução de Sinais/efeitos dos fármacos , Adipocinas/genética , Adipocinas/metabolismo , Animais , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrose , Cardiopatias/etiologia , Cardiopatias/prevenção & controle , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Proteína Smad2/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Biotechnol Bioeng ; 118(6): 2202-2219, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33624859

RESUMO

Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293-E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme-linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in-depth characterization of SARS-CoV-2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID-19 serologic tests.


Assuntos
Antígenos Virais/biossíntese , Glicosilação , Glicoproteína da Espícula de Coronavírus/biossíntese , Temperatura Baixa , Ensaio de Imunoadsorção Enzimática/normas , Congelamento , Células HEK293 , Humanos , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/normas , SARS-CoV-2 , Testes Sorológicos/normas , Glicoproteína da Espícula de Coronavírus/normas
15.
Food Chem ; 350: 129212, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33609939

RESUMO

A novel alkaline cold-active phospholipase C (PLC) gene (AoPC) from Aspergillus oryzae was cloned. AoPC exhibited the highest sequence similarity of 32.5% with that of a PLC from Arabidopsis thaliana. The gene was co-expressed in Pichia pastoris with molecular chaperone PDI (protein disulfide isomerases), and the highest PLC activity of 82, 782 U mL-1 was achieved in a 5-L fermentor. The recombinant enzyme (AoPC) was most active at pH 8.0 and 25 °C, respectively, and it was stable over a broad pH range of 4.5-9.0 and up to 40 °C. It is the first fungal alkaline PLC. The application of AoPC (with 25% citric acid, w/w) in oil degumming process significantly reduced the phosphorus of crude soybean oil by 93.3% to a commercially acceptable level (<10 mg kg-1). Therefore, the relatively high yield and excellent properties of AoPC may possess it great potential in crude oil refining industry.


Assuntos
Aspergillus oryzae/enzimologia , Temperatura Baixa , Engenharia Genética/métodos , Chaperonas Moleculares/genética , Petróleo/análise , Fosfolipases Tipo C/biossíntese , Fosfolipases Tipo C/metabolismo , Clonagem Molecular , Expressão Gênica , Concentração de Íons de Hidrogênio , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fosfolipases Tipo C/genética
16.
Arch Microbiol ; 203(4): 1281-1292, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33481073

RESUMO

Amylase is amongst the most indispensable enzymes that have a large number of applications in laboratories and industries. Mostly, α-amylase is synthesized from microbes such as bacteria, fungi and yeast. Due to the high demand for α-amylase, its synthesis can be enhanced using recombinant DNA technology, different fermentation methods, less expensive and good carbon and nitrogen sources, and optimizing the various parameters during fermentation, e.g., temperature, pH and fermentation duration. Various methods are used to measure the production and activity of synthesized α-amylase like iodine, DNS, NS and dextrinizing methods. The activity of crude α-amylase can be elevated to the maximum level by optimizing the temperature and pH. Some metals also interact with α-amylase and increase its activity like K+, Na+, Mg2+ and Ca2+. Some industries such as starch conversion, food, detergent, paper, textile industries and fuel alcohol production extensively utilize α-amylase for their various purposes.


Assuntos
alfa-Amilases/biossíntese , Bactérias/metabolismo , Carbono/metabolismo , Fermentação , Fungos/metabolismo , Concentração de Íons de Hidrogênio , Indústrias , Metais , Nitrogênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , alfa-Amilases/genética , alfa-Amilases/metabolismo
17.
Molecules ; 26(3)2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33499126

RESUMO

The importance of bioprocesses has increased in recent decades, as they are considered to be more sustainable than chemical processes in many cases. E factors can be used to assess the sustainability of processes. However, it is noticeable that the contribution of enzyme synthesis and purification is mostly neglected. We, therefore, determined the E factors for the production and purification of 10 g enzymes. The calculated complete E factor including required waste and water is 37,835 gwaste·genzyme-1. This result demonstrates that the contribution of enzyme production and purification should not be neglected for sustainability assessment of bioprocesses.


Assuntos
Enzimas/biossíntese , Enzimas/isolamento & purificação , Química Verde/métodos , Biocatálise , Bioengenharia , Reatores Biológicos , Engenharia Química , Indústria Farmacêutica , Meio Ambiente , Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Resíduos Industriais , Nucleotidiltransferases/biossíntese , Nucleotidiltransferases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação
18.
Food Chem ; 346: 128953, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33412487

RESUMO

Tartary buckwheat is widely accepted as its nutritionalvalue. Some allergic reactions hinder its utilization. This research focused on evaluating the core epitope of 16 kDa allergen (Fag t 2) in tartary buckwheat. Six B- and seven T cell epitopes of Fag t 2 were predicted, and six B cell epitope-mutants were expressed in Pichia pastoris. Bioinformatics analysis and SDS-PAGE demonstrated that the molecular weight, isoelectric point and spatial structures of six mutant allergens were similar with Fag t 2, with the same signal peptide sequences and α-amylase inhibitor domain. There was no significant change in mutants' spatial conformation confirmed by Circular Dichroism. The position K132N and peptides at 108-117 and 132-141 were the core B- and T cell epitopes of Fag t 2 confirmed by competitive inhibition ELISA and dot blot. This result was of great significance on the study of allergen epitopes in prevention and treatment of hypersensitivity.


Assuntos
Alérgenos/imunologia , Epitopos/imunologia , Fagopyrum/metabolismo , Alérgenos/química , Alérgenos/genética , Alérgenos/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/metabolismo , Mutagênese Sítio-Dirigida , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
19.
Protein Expr Purif ; 177: 105746, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32916300

RESUMO

Periplasmic expression of recombinant proteins ensures the production of biologically active proteins in a correctly folded state with several key advantages. This research focused on the in-frame cloning of rhIL-15 in pET-20 (+) vector with pelB-leader sequence to direct the protein to the bacterial periplasm. The target construct periplasmic expression was evaluated in four strains, BL21 (DE3), BL21 (DE3) pLysS, Rosetta 2 (DE3) and Rosetta-gami 2 (DE3). Soluble periplasmic expression of IL-15 was highest in Rosetta-gami 2 (DE3) followed by Rossetta 2 (DE3) whereas negligible expression was observed with rest of two expression host. Best expression clone was selected for purification by dye ligand affinity chromatography. Purified rhIL-15 was characterized by SDS-PAGE, Western blotting and SEC-HPLC. This is the first report of functional recombinant human interleukin-15 being expressed and purified with yield of 120 mg/L in the periplasmic space of E. coli.


Assuntos
Clonagem Molecular/métodos , Interleucina-15/genética , Periplasma/genética , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Interleucina-15/biossíntese , Interleucina-15/farmacologia , Camundongos , Periplasma/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Solubilidade , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
20.
Biochim Biophys Acta Biomembr ; 1863(1): 183441, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32810489

RESUMO

G protein coupled receptors (GPCRs) function as guanine nucleotide exchange factors (GEFs) at heterotrimeric G proteins, and conduct this role embedded in a lipid bilayer. Detergents are widely used to solubilise GPCRs for structural and biophysical analysis, but are poor mimics of the lipid bilayer and may be deleterious to protein function. Amphipathic polymers have emerged as promising alternatives to detergents, which maintain a lipid environment around a membrane protein during purification. Of these polymers, the polymethacrylate (PMA) polymers have potential advantages over the most popular styrene maleic acid (SMA) polymer, but to date have not been applied to purification of membrane proteins. Here we use a class A GPCR, neurotensin receptor 1 (NTSR1), to explore detergent-free purification using PMA. By using an NTSR1-eGFP fusion protein expressed in Sf9 cells, a range of solubilisation conditions were screened, demonstrating the importance of solubilisation temperature, pH, NaCl concentration and the relative amounts of polymer and membrane sample. PMA-solubilised NTSR1 displayed compatibility with standard purification protocols and millimolar divalent cation concentrations. Moreover, the receptor in PMA discs showed stimulation of both Gq and Gi1 heterotrimers to an extent that was greater than that for the detergent-solubilised receptor. PMA therefore represents a viable alternative to SMA for membrane protein purification and has a potentially broad utility in studying GPCRs and other membrane proteins.


Assuntos
Ácidos Polimetacrílicos/química , Receptores de Neurotensina , Detergentes/química , Humanos , Receptores de Neurotensina/biossíntese , Receptores de Neurotensina/química , Receptores de Neurotensina/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Solubilidade
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