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1.
Talanta ; 201: 397-405, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122440

RESUMO

This article reports the identification, engineering and characterisation of recombinant single chain variable fragment (scFv) antibody against Zearalenone (ZEN), an oestrogenic mycotoxin, using phage display antibody technology. To increase the chance of obtaining clones that can bind to free toxin, the conjugated proteins of the target antigen, i.e. bovine serum albumin ZEN-BSA and ovalbumin ZEN-OVA, were switched during the biopanning. One phage-displayed scFv clone specific to free ZEN, designated yZEN2A8, could be isolated. The gene encoding the yZEN2A8 scFv was sub-cloned into the pET-21d (+) and pKP300 delta III vectors to generate the recombinant scFv and scFv-AP antibody formats, respectively. After ELISA optimisation by checkerboard titration, the sensitivities of the recombinant yZEN2A8 scFv antibody and scFv-AP fusion were improved approx. 2 and 60 folds, respectively. Competitive ELISA indicated that the median inhibition concentration (IC50) of recombinant yZEN2A8 scFv antibody and scFv-AP fusion after ELISA optimisation were 90 and 14 ng mL-1, with a limit of detection (LOD) of 20 and 2 ng mL-1, respectively. No cross-reactivity to other common mycotoxins was observed. Homology modelling illustrated specific binding of the recombinant antibody to ZEN and demonstrated the role of complementary determining regions (CDRs) of both the variable heavy and light chains in antibody-antigen interactions. Efficient application of scFv-AP for the detection of ZEN contamination in corns and wheat samples were investigated for the first time. The antibody in the form of scFv-AP can be used as a prototype for the development of a convenient reagent for the detection of ZEN contamination in various format, including biosensor-based.


Assuntos
Ração Animal/análise , Contaminação de Alimentos/análise , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/imunologia , Zearalenona/análise , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Técnicas de Visualização da Superfície Celular/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Humanos , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Ligação Proteica , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Triticum/química , Zea mays/química , Zearalenona/imunologia , Zearalenona/metabolismo
2.
Chem Biol Interact ; 306: 89-95, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30986387

RESUMO

Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a ~2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an N-terminal fusion protein, Fc(M3)-(PAPAP)2-CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP)2-CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105 ±â€¯7 h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP)2-CocH3 has a ~21-fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP)2-CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction.


Assuntos
Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Fragmentos Fc das Imunoglobulinas/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Animais , Células CHO , Hidrolases de Éster Carboxílico/genética , Cricetulus , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética
3.
Mol Med Rep ; 19(5): 4514-4522, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30942410

RESUMO

Thyroid stimulating hormone (TSH) consists of an α­subunit and a unique ß­subunit. The first in­frame TSHß splice variant produced by the cells of immune system was identified in 2009. The TSHß splice variant and native TSHß exhibit different expression profiles, and research has been conducted to elucidate the role of the TSHß splice variant in different diseases. However, understanding of the fundamental physiological characteristics of the TSHß splice variant is currently limited. To verify whether the TSHß splice variant has the potential to induce thyroid follicular cells to synthesize thyroid hormone, in vivo and in vitro stimulation experiments were conducted in the present study. A total of 60 C57BL/6 mice were divided into control­, 5 and 10 µg TSHß splice variant­treated groups at random. Mice were sacrificed at 0.5, 1 and 4 h after intraperitoneal injection, and serum levels of tri­iodothyronine (T3) and thyroxine (T4) were determined using a radioimmunoassay. Thyroid follicular cells were isolated from the thyroids of mice, and stimulated with 2 µg/ml TSHß splice variant. Supernatants were collected, and the levels of T3 and T4 were detected. The protein expression levels of the sodium­iodide symporter, thyroperoxidase and thyroglobulin in thyroid follicular cells were quantified using western blot analysis. To verify whether the TSHß splice variant expression was regulated by the hypothalamus­pituitary­thyroid (HPT) axis, similar to native TSHß, a total of 60 C57BL/6 mice were equally divided into control, 2 mg/kg T3 intraperitoneal injection and 0.05 mg/kg thyroid­releasing hormone intraperitoneal injection groups at random. Mice were sacrificed at 1 and 4 h after injection. Alterations in the expression of the TSHß splice variant in the pituitary, thyroid, peripheral blood leukocytes and spleen tissues were detected using western blot analysis. The present study demonstrated that the TSHß splice variant is not regulated by the HPT axis and may affect thyroid hormone synthesis. Modifications in the expression of the TSHß splice variant may occur in a uniquely regulated manner to provide peripheral immunological compartments with a source of activated cells, particularly under immune stress.


Assuntos
Hormônios Tireóideos/biossíntese , Tireotropina Subunidade beta/genética , Animais , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Processamento de RNA , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Simportadores/metabolismo , Tireoglobulina/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Tireotropina Subunidade beta/metabolismo , Tiroxina/sangue , Tri-Iodotironina/sangue
4.
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
5.
Bioengineered ; 10(1): 87-97, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30957636

RESUMO

Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor α1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)20, was engineered either at the N- or C-terminal end of AAT. The (AP)20 tag substantially increased the secreted yields of the recombinant AAT up to 34.7 mg/L. However, the (AP)20-tagged AAT products were frequently subjected to proteolytic processing. The intact AAT-(AP)20 along with some of the truncated AAT domains exhibited desired biological activity in inhibiting elastase. The results from this research demonstrated that the designer (AP)20 module engineered in BY-2 cells could function as a molecular carrier to substantially enhance the secreted yields of the recombinant AAT.


Assuntos
Elastase Pancreática/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Proteínas Secretadas Inibidoras de Proteinases/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tabaco/genética , alfa 1-Antitripsina/biossíntese , Sequência de Bases , Técnicas de Cultura de Células , Dipeptídeos/genética , Dipeptídeos/metabolismo , Expressão Gênica , Glicosilação , Humanos , Elastase Pancreática/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Células Vegetais/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Tabaco/citologia , Tabaco/metabolismo , Transformação Genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/farmacologia
6.
Molecules ; 24(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991754

RESUMO

Cell surface display systems for immobilization of peptides and proteins on the surface of cells have various applications, such as vaccine generation, protein engineering, bio-conversion and bio-adsorption. Though plenty of methods have been established in terms of traditional yeast surface display systems, the development of a universal display method with high efficiency remains a challenge. Here we report an indirect yeast surface display method by anchoring Im7 proteins on the surface of P. pastoris, achieving highly efficient display of target proteins, including fluorescence proteins (sfGFP and mCherry) or enzymes (human Arginase I), with a CL7 fusion tag through the ultra-high-affinity interaction between Im7 and CL7. This indirect P. pastoris surface display approach is highly efficient and provides a robust platform for displaying biomolecules.


Assuntos
Proteínas Fúngicas , Expressão Gênica , Pichia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
7.
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
8.
Cell Physiol Biochem ; 52(4): 681-695, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30921507

RESUMO

BACKGROUND/AIMS: Oxidative modifications of low-density lipoprotein (ox-LDL) play a key role in initial steps of atheroprogression possibly via specific scavenger receptors on inflammatory and endothelial cells. Amongst others, CD68 might play a crucial role in this leading to fatty streak formation. METHODS: Different CD68-Fc fusion proteins were cloned, expressed and tested in vitro for their oxLDL binding properties as a decoy for endogenous oxLDL. Physiological functions were tested in foam cell assays with human monocytes in culture and by binding oxLDL from human blood. The best suited candidate FcIgG2-FL-CD68 was injected twice weekly in LDL receptor and ApoBec deficient mice (LDLR-/-/Apobec-/-), and the oxLDL content was measured in peripheral blood, in different cell types of the spleen and aortic wall by specific oxLDL antibodies using flow cytometry. RESULTS: Different variants of the CD68-Fc bound to copper-oxided LDL (oxLDL), LDL and to a lesser extent HDL with different efficacy in an ELISA based binding assay in vitro. Native oxLDL content in human blood derived from patients with extended atherosclerosis was reduced after passage through a specific protein G column conjugated with the different CD68-Fc fusion proteins. Foam cell formation from human peripheral blood monocyte-platelet co-culture was reduced by the most effective CD68-Fc fusion proteins. oxLDL was not increased in the blood but markedly increased in the vessel wall from LDLR-/-/Apobec-/- mice at an early stage of atherosclerosis. Platelet-like cells in the vessel well contributed most to the increase in tissue oxLDL. FcIgG2-FL-CD68, reduced oxLDL content of aortic vessel wall cells from LDLR-/-/Apobec-/- mice. However a tissue specific reduction on the oxLDL content in peripheral blood, the spleen or cells from the aortic vessel by FcIgG2-FL-CD68 could not be shown. CONCLUSION: Platelets contribute to increased tissue oxLDL in the aortic wall but not in peripheral blood. CD68 seems to play a role in the oxLDL metabolism in the vessel wall at early stages of atherosclerosis. FcIgG2-FL-CD68 could serve as a novel therapeutic option to modify the oxLDL content in the vessel wall.


Assuntos
Desaminase APOBEC-1/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Plaquetas/metabolismo , Lipoproteínas LDL/genética , Desaminase APOBEC-1/deficiência , Animais , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Plaquetas/citologia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Modelos Animais de Doenças , Células Espumosas/citologia , Células Espumosas/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/análise , Lipoproteínas LDL/deficiência , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação
9.
Biomed Res Int ; 2019: 8010635, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30915359

RESUMO

ß-Galactosidase (E.C.3.2.1.23) catalyzes the hydrolysis of lactose into glucose and galactose and the synthesis of galacto-oligosaccharides as well. The ß-galactosidases from bacteria, especially lactobacilli, and yeast have neutral pH and are much more likely to be developed as food additives. However, the challenges of cumbersome purification, product toxicity, and low yield in protein production have limited the commercialization of many excellent candidates. In this study, we identified a ß-galactosidase gene (bg42-106) in Bifidobacterium animalis ACCC05790 and expressed the gene product in Escherichia coli BL21(DE3) and Pichia pastoris GS115, respectively. The recombinant bG42-106 purified from E. coli cells was found to be optimally active at pH 6.0 and 60°C and had excellent stability over a wide pH range (5.0-8.0) and at high temperature (60°C). The specific activity of bG42-106 reached up to 2351 U/mg under optimal conditions. The galacto-oligosaccharide yield was 24.45 g/L after incubation with bG42-106 at 60°C for 2 h. When recombinant bG42-106 was expressed in Pichia pastoris GS115, it was found in the culture medium but only at a concentration of 1.73 U/ml. To increase its production, three strategies were employed, including codon optimization, disulfide formation, and fusion with a Cherry tag, with Cherry-tag fusion being most effective. The culture medium of P. pastoris that expressed Cherry-tagged bG42-106 contained 24.4 U/mL of ß-galactosidase activity, which is 14-fold greater than that produced by culture of P. pastoris harboring wild-type bG42-106.


Assuntos
Proteínas de Bactérias , Bifidobacterium animalis/enzimologia , Bifidobacterium animalis/genética , Pichia , Proteínas Recombinantes de Fusão , beta-Galactosidase , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , beta-Galactosidase/biossíntese , beta-Galactosidase/genética , beta-Galactosidase/isolamento & purificação
10.
Appl Microbiol Biotechnol ; 103(7): 3061-3071, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30783720

RESUMO

A simple and stable immobilization of a laccase from Pleurotus ostreatus was obtained through genetic fusion with a self-assembling and adhesive class I hydrophobin. The chimera protein was expressed in Pichia pastoris and secreted into the culture medium. The crude culture supernatant was directly used for coatings of polystyrene multi-well plates without additional treatments, a procedure that resulted in a less time-consuming and chemicals reduction. Furthermore, the gene fusion yielded a positive effect with respect to the wild-type recombinant enzyme in terms of both immobilization and stability. The multi-well plate with the immobilized chimera was used to develop an optical biosensor to monitor two phenolic compounds: L-DOPA ((S)-2-amino-3-(3,4-dihydroxyphenyl) propanoic acid) and caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid); the estimation of which is a matter of interest in the pharmaceutics and food field. The method was based on the use of the analytes as competing inhibitors of the laccase-mediated ABTS oxidation. The main advantages of the developed biosensor are the ease of preparation, the use of small sample volumes, and the simultaneous analysis of multiple samples on a single platform.


Assuntos
Técnicas Biossensoriais , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Pleurotus/enzimologia , Ácidos Cafeicos/metabolismo , Clonagem Molecular , Meios de Cultura/química , Enzimas Imobilizadas/biossíntese , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Lacase/genética , Levodopa/metabolismo , Oxirredução , Pichia/genética , Poliestirenos , Proteínas Recombinantes de Fusão/biossíntese
11.
Food Chem ; 285: 204-212, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30797336

RESUMO

Prebiotic fructooligosaccharides (FOS) are currently obtained by enzymatic reaction with fructosyltransferases (FTFs) using sucrose as both donor and acceptor. In these reactions glucose results as the most abundant by-product, arising from each fructosyl transfer event and, together with fructose, because of the inherent hydrolytic activity of the FTFs. As FOS are mainly used as prebiotic in nutraceutical foods, the reduction or total elimination of monosaccharides is required. In this work the selective elimination of monosaccharides from a synthetic FOS mixture was achieved through the selective complexation of glucose and fructose with phenyl boronic acid (PBAc) followed by ethyl-acetate extraction. The process was applied to a complex mixture of FOS obtained in an enzymatic synthesis reaction containing 40% glucose, 15.8% fructose and 35% of FOS, elimination of the sugars was achieved through 3:1 molar reactions, resulting in a levan-type FOS product with 97% purity.


Assuntos
Ácidos Borônicos/metabolismo , Monossacarídeos/metabolismo , Oligossacarídeos/isolamento & purificação , Acetatos/química , Ácidos Borônicos/química , Cromatografia em Camada Delgada , Escherichia coli/metabolismo , Frutose/química , Glucose/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Extração Líquido-Líquido , Monossacarídeos/química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Prebióticos/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética
12.
Food Chem ; 282: 101-108, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711093

RESUMO

A one-step method to immobilize xylanase onto cellulosic material by fusion of expansin from Bacillus subtilis to xylanase LC9 without the requirement of prior purification of enzyme has been developed. Fusion enzyme EXLX-R2-XYN was specifically adsorbed onto corncob residue with high loading capacity due to bio-affinity adsorption of expansin onto cellulose. The immobilization yield was close to 100%, with a recovered activity of 82.4%. The immobilized EXLX-R2-XYN retained 45.3% of its activity after incubation at 70 °C for 3 h, whereas only 16.3% of the activity was left in free form under the same conditions. The conversion yield of XOS by using immobilized EXLX-R2-XYN reached up to 515 mg/g xylan from 2% corncob extracted xylan, which was higher than that of the free enzyme. The hydrolysis products were mainly xylobiose (57.5%) and xylotriose (38.4%), without undesirable xylose production. After five cycles of hydrolysis, more than 70% of conversion was obtained.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Glucuronatos/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Bactérias/genética , Cromatografia de Afinidade , Dissacarídeos/metabolismo , Endo-1,4-beta-Xilanases/genética , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glucuronatos/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Oligossacarídeos/isolamento & purificação , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Reciclagem , Temperatura Ambiente , Trissacarídeos/metabolismo
13.
Microb Cell Fact ; 18(1): 31, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30732606

RESUMO

BACKGROUND: Heterologous gene expression is well established for various prokaryotic model systems. However, low yield, incorrect folding and instability still impede the production of soluble, bioactive proteins. To improve protein production with the Gram-positive host Bacillus subtilis, a secretory expression system was designed that enhances translocation, folding and stability of heterologous proteins, and simplifies purification. Based on the theta-replication plasmid pHT01, a B. subtilis secretory expression vector was constructed that encodes a fusion protein consisting of a signal peptide and a StrepII-tag linked to a SUMO-tag serving as a folding catalyst. The gene of a protein of interest can be translationally fused to the SUMO cassette and an additional 6xHis-tag encoding region. In order to maximize secretory expression of the construct by fitting the signal peptide to the StrepII-SUMO part of the fusion protein, a B. subtilis signal-peptide library was screened with the Escherichia coli alkaline phosphatase PhoA as a reporter. RESULTS: The YoaW signal peptide-encoding region (SPyoaW) was identified with highest secretory expression capacity in context with the StrepII-SUMO-tag fusion in a B. subtilis eightfold extracellular protease deletion strain. PhoA activity and fusion protein production was elevated by a factor of approximately five when compared to an α-amylase (AmyQ) signal peptide construct. Replacement of PhoA with a single-chain variable fragment antibody specific for GFP or the B. amyloliquefaciens RNase barnase, respectively, resulted in a similar enhancement of secretory expression, demonstrating universality of the YoaW signal peptide-StrepII-SUMO encoding cassette for secretory expression in B. subtilis. Optimisation of codon usage and culture conditions further increased GFP-specific scFv fusion-protein production, and a simple affinity purification strategy from culture supernatant with removal of the StrepII-SUMO-tag by SenP-processing yielded 4 mg of pure, soluble and active GFP-specific scFv from 1 l of culture under standard laboratory conditions. CONCLUSIONS: The new expression system employing a YoaW signal peptide-StrepII-SUMO fusion will simplify secretory protein production and purification with B. subtilis. It can obviate the need for time consuming individual signal-peptide fitting to maximize yield for many different heterologous proteins of interest.


Assuntos
Bacillus subtilis/genética , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/biossíntese , Fosfatase Alcalina/metabolismo , Bacillus subtilis/química , Escherichia coli/enzimologia , Expressão Gênica , Biblioteca de Peptídeos , Plasmídeos/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética
14.
Appl Microbiol Biotechnol ; 103(5): 2205-2216, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30610290

RESUMO

The Escherichia coli (E. coli) expression system has been widely used to produce recombinant proteins. However, in some heterologous expressions, there are still difficulties in large-scale production. The use of fusion partners is one of the strategies for improving the expression levels of proteins in E. coli host. Here, we demonstrate a novel fusion element, the NT11-tag, which enhances protein expression. The NT11-tag was derived from the first 11 amino acid residues within the N-terminal N-half domain of a duplicated carbonic anhydrase (dCA) from Dunaliella species. Previously, we have found that the tag improves expression of the C-half domain of dCA when linked to its N-terminus. To verify its use as a protein production enhancer tag, two kinds of CAs derived from Hahella chejuensis (Hc-CA) and Thermovibrio ammonifican (Ta-CA) and the yellow fluorescent protein (YFP) were used as model proteins to measure their increased expression upon fusion with the NT11-tag. The NT11-tag amplified protein expression in E. coli by 6.9- and 7.6-fold for Ta-CA and YFP, respectively. Moreover, the tag also enhanced the soluble expression of Hc-CA, Ta-CA, and YFP by 1.7-, 5.0-, and 3.2-fold, respectively. Furthermore, protein yield was increased without inhibiting protein function. These results indicate that the use of the NT11-tag is a promising method for improving protein production in E. coli.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Escherichia coli/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica/genética , Proteínas Luminescentes/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas de Bactérias/genética , Anidrases Carbônicas/genética , Proteínas de Escherichia coli/genética , Proteínas Luminescentes/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética
15.
Appl Microbiol Biotechnol ; 103(5): 2229-2241, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30631897

RESUMO

L-Amino acid oxidases (LAAOs) are flavoproteins, which use oxygen to deaminate L-amino acids and produce the corresponding α-keto acids, ammonia, and hydrogen peroxide. Here we describe the heterologous expression of LAAO4 from the fungus Hebeloma cylindrosporum without signal sequence as fusion protein with a 6His tag in Escherichia coli and its purification. 6His-hcLAAO4 could be activated by exposure to acidic pH, the detergent sodium dodecyl sulfate, or freezing. The enzyme converted 14 proteinogenic L-amino acids with L-glutamine, L-leucine, L-methionine, L-phenylalanine, L-tyrosine, and L-lysine being the best substrates. Methyl esters of these L-amino acids were also accepted. Even ethyl esters were converted but with lower activity. Km values were below 1 mM and vmax values between 19 and 39 U mg-1 for the best substrates with the acid-activated enzyme. The information for an N-terminal aldehyde tag was added to the coding sequence. Co-expressed formylglycine-generating enzyme was used to convert a cysteine residue in the aldehyde tag to a Cα-formylglycine residue. The aldehyde tag did not change the properties of the enzyme. Purified Ald-6His-hcLAAO4 was covalently bound to a hexylamine resin via the Cα-formylglycine residue. The immobilized enzyme could be reused repeatedly to generate phenylpyruvate from L-phenylalanine with a total turnover number of 17,600 and was stable for over 40 days at 25 °C.


Assuntos
Enzimas Imobilizadas/metabolismo , Hebeloma/enzimologia , L-Aminoácido Oxidase/metabolismo , Fenilalanina/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , L-Aminoácido Oxidase/genética , Proteínas Recombinantes de Fusão/genética
16.
Appl Microbiol Biotechnol ; 103(6): 2809-2820, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30666362

RESUMO

Enzymes could act as a useful tool for environmental bioremediation. Arsenic (As) biomethylation, which can convert highly toxic arsenite [As(III)] into low-toxic volatile trimethylarsine, is considered to be an effective strategy for As removal from contaminated environments. As(III) S-adenosylmethyltransferase (ArsM) is a key enzyme for As methylation; its properties and preparation are crucial for its wide application. Currently, ArsM is usually purified as a His-tag fusion protein restricting widespread use due to high costs. In this study, to greatly reduce the cost and simplify the ArsM preparation process, an Elastin-like polypeptide (ELP) tag was introduced to construct an engineered Escherichia coli (ArsM-ELP). Consequently, a cost-effective and simple non-chromatographic purification approach could be used for ArsM purification. The enzymatic properties of ArsM-ELP were systematically investigated. The results showed that the As methylation rate of purified ArsM-ELP (> 35.49%) was higher than that of E. coli (ArsM-ELP) (> 10.39%) when exposed to 25 µmol/L and 100 µmol/L As(III), respectively. The purified ArsM-ELP was obtained after three round inverse transition cycling treatment in 2.0 mol/L NaCl at 32 °C for 10 min with the yield reaching more than 9.6% of the total protein. The optimal reaction temperature, pH, and time of ArsM-ELP were 30 °C, 7.5 and 30 min, respectively. The enzyme activity was maintained at over 50% at 45 °C for 12 h. The enzyme specific activity was 438.8 ± 2.1 U/µmol. ArsM-ELP had high selectivity for As(III). 2-Mercaptoethanol could promote enzyme activity, whereas SDS, EDTA, Fe2+, and Cu2+ inhibited enzyme activity, and Mg2+, Zn2+, Ca2+, and K+ had no significant effects on it.


Assuntos
Arsênico/metabolismo , Elastina/biossíntese , Escherichia coli/genética , Metiltransferases/biossíntese , Biodegradação Ambiental , Elastina/genética , Escherichia coli/enzimologia , Engenharia Genética , Metilação , Metiltransferases/genética , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , S-Adenosilmetionina/metabolismo , Temperatura Ambiente
17.
MAbs ; 11(3): 559-568, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30694096

RESUMO

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.


Assuntos
Anticorpos Monoclonais Murinos , Antígenos , Imunoglobulina A , Imunoglobulina G , Região Variável de Imunoglobulina , Proteínas Recombinantes de Fusão , Animais , Anticorpos Monoclonais Murinos/biossíntese , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/genética , Antígenos/biossíntese , Antígenos/química , Antígenos/genética , Células CHO , Cricetulus , Imunoglobulina A/biossíntese , Imunoglobulina A/química , Imunoglobulina A/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Camundongos , Peptídeos/química , Peptídeos/genética , Proteólise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Suínos
18.
PLoS One ; 13(12): e0208579, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30566445

RESUMO

Exogenous vascular endothelial growth factor (VEGF) accelerates compensatory lung growth (CLG) in mice after unilateral pneumonectomy. In this study, we unexpectedly discovered a method to enhance CLG with a VEGF inhibitor, soluble VEGFR1. Eight-week-old C57BL/6 male mice underwent left pneumonectomy, followed by daily intraperitoneal (ip) injection of either saline (control) or 20 µg/kg of VEGFR1-Fc. On post-operative day (POD) 4, mice underwent pulmonary function tests (PFT) and lungs were harvested for volume measurement and analyses of the VEGF signaling pathway. To investigate the role of hypoxia in mediating the effects of VEGFR1, experiments were repeated with concurrent administration of PT-2385, an inhibitor of hypoxia-induced factor (HIF)2α, via orogastric gavage at 10 mg/kg every 12 hours for 4 days. We found that VEGFR1-treated mice had increased total lung capacity (P = 0.006), pulmonary compliance (P = 0.03), and post-euthanasia lung volume (P = 0.049) compared to control mice. VEGFR1 treatment increased pulmonary levels of VEGF (P = 0.008) and VEGFR2 (P = 0.01). It also stimulated endothelial proliferation (P < 0.0001) and enhanced pulmonary surfactant production (P = 0.03). The addition of PT-2385 abolished the increase in lung volume and endothelial proliferation in response to VEGFR1. By paradoxically stimulating angiogenesis and enhancing lung growth, VEGFR1 could represent a new treatment strategy for neonatal lung diseases characterized by dysfunction of the HIF-VEGF pathway.


Assuntos
Pulmão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Relação Dose-Resposta a Droga , Meia-Vida , Pulmão/crescimento & desenvolvimento , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Pneumonectomia , Proteínas Recombinantes de Fusão/biossíntese , Testes de Função Respiratória , Transdução de Sinais/efeitos dos fármacos , Tensoativos/metabolismo , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
19.
J Photochem Photobiol B ; 185: 66-72, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29870960

RESUMO

Firefly luciferase (Fluc) has been widely used as a bioluminescent monitor. The ATP linear correlation and exogenous luciferin requirement make it useful in most of current imaging systems. However, the utility of this reporter was still limited by the intensity and decay of the luminescent signal, and the active site and structure of enzyme including the relevant substrate channeling region. This study demonstrated a novel construction of bifunctional enzyme system to improve the luminescence generation of firefly luciferase, by bringing in a luciferin-regenerating enzyme (LRE) fusion expressed to the C terminal of luciferase, between which were connected with peptide linker. The fusion protein constructed with typical type of linker, rigid linker (EAAAK) and flexible linker (GGGGS), were analyzed comparing with the unlinked free enzyme. In vivo and in vitro assessment of the bioluminescence intensity and decaying rate to the series of Fluc-LRE enzyme complex were assayed. The fInding demonstrated that the presence of LRE remarkably enhance the generation of luminescence and remained significant stronger signal than that of the control, and the peptide-linked dual enzyme present more stability and continuation on the signal generation and lower decaying rate on signal recession, especially at low dose of Fluc injection. With the advantage of luminescence intensity and reaction period, the peptide mediated fusion expressed LRE may expand the application of Firefly luciferase on bioluminescence imaging.


Assuntos
Luciferina de Vaga-Lumes/metabolismo , Luciferases de Vaga-Lume/metabolismo , Sequência de Aminoácidos , Animais , Cisteína/química , Cisteína/metabolismo , Escherichia coli/metabolismo , Luciferina de Vaga-Lumes/química , Cinética , Luciferases de Vaga-Lume/genética , Medições Luminescentes , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética
20.
BMB Rep ; 51(7): 362-367, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29936932

RESUMO

A major feature of type 1 diabetes mellitus (T1DM) is hyperglycemia and dysfunction of pancreatic ß-cells. In a previous study, we have shown that Tat-DJ-1 protein inhibits pancreatic RINm5F ß-cell death caused by oxidative stress. In this study, we examined effects of Tat-DJ-1 protein on streptozotocin (STZ)-induced diabetic mice. Wild type (WT) Tat-DJ-1 protein transduced into pancreas where it markedly inhibited pancreatic ß-cell destruction and regulated levels of serum parameters including insulin, alkaline phosphatase (ALP), and free fatty acid (FFA) secretion. In addition, transduced WT Tat-DJ-1 protein significantly inhibited the activation of NF-κB and MAPK (ERK and p38) expression as well as expression of COX-2 and iNOS in STZ exposed pancreas. In contrast, treatment with C106A mutant Tat-DJ-1 protein showed no protective effects. Collectively, our results indicate that WT Tat-DJ-1 protein can significantly ameliorate pancreatic tissues in STZ-induced diabetes in mice. [BMB Reports 2018; 51(7): 362-367].


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Produtos do Gene tat/genética , Substâncias Protetoras/uso terapêutico , Proteína Desglicase DJ-1/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Fosfatase Alcalina/sangue , Animais , Ciclo-Oxigenase 2/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ácidos Graxos não Esterificados/sangue , Insulina/sangue , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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