RESUMO
Recombinant multiepitope proteins (RMPs) are a promising alternative for application in diagnostic tests and, given their wide application in the most diverse diseases, this review article aims to survey the use of these antigens for diagnosis, as well as discuss the main points surrounding these antigens. RMPs usually consisting of linear, immunodominant, and phylogenetically conserved epitopes, has been applied in the experimental diagnosis of various human and animal diseases, such as leishmaniasis, brucellosis, cysticercosis, Chagas disease, hepatitis, leptospirosis, leprosy, filariasis, schistosomiasis, dengue, and COVID-19. The synthetic genes for these epitopes are joined to code a single RMP, either with spacers or fused, with different biochemical properties. The epitopes' high density within the RMPs contributes to a high degree of sensitivity and specificity. The RMPs can also sidestep the need for multiple peptide synthesis or multiple recombinant proteins, reducing costs and enhancing the standardization conditions for immunoassays. Methods such as bioinformatics and circular dichroism have been widely applied in the development of new RMPs, helping to guide their construction and better understand their structure. Several RMPs have been expressed, mainly using the Escherichia coli expression system, highlighting the importance of these cells in the biotechnological field. In fact, technological advances in this area, offering a wide range of different strains to be used, make these cells the most widely used expression platform. RMPs have been experimentally used to diagnose a broad range of illnesses in the laboratory, suggesting they could also be useful for accurate diagnoses commercially. On this point, the RMP method offers a tempting substitute for the production of promising antigens used to assemble commercial diagnostic kits.
Assuntos
Epitopos , Escherichia coli , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Humanos , Epitopos/imunologia , Epitopos/genética , Testes Imunológicos/métodos , Animais , COVID-19/diagnósticoRESUMO
The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a ß-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.
Assuntos
Bacillus subtilis , Celulase , Papel , Proteínas Recombinantes , Águas Residuárias , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/isolamento & purificação , Águas Residuárias/microbiologia , Águas Residuárias/química , Celulase/genética , Celulase/química , Celulase/biossíntese , Celulase/isolamento & purificação , Celulase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Clonagem Molecular , Expressão GênicaRESUMO
Improving the diagnostic technology used to detect tegumentary leishmaniasis (TL) is essential in view of it being a widespread, often neglected tropical disease, with cases reported from the Southern United States to Northern Argentina. Recombinant proteins, recombinant multiepitope proteins, and synthetic peptides have been extensively researched and used in disease diagnosis. One of the benefits of applying these antigens is a measurable increase in sensitivity and specificity, which improves test accuracy. The present review aims to describe the use of these antigens and their diagnostic effectiveness. With that in mind, a bibliographic survey was conducted on the PudMed platform using the search terms "tegumentary leishmaniasis" AND "diagno", revealing that recombinant proteins have been described and evaluated for their value in TL diagnosis since the 1990s. However, there was a spike in the number of publications using all of the antigens between 2013 and 2022, confirming an expansion in research efforts to improve diagnosis. Moreover, all of the studies involving different antigens had promising results, including improved sensitivity and specificity. These data recognize the importance of doing research with new technologies focused on developing quick, more effective diagnostic kits as early diagnosis facilitates treatment.
Assuntos
Antígenos de Protozoários , Leishmaniose Cutânea , Proteínas Recombinantes , Antígenos de Protozoários/imunologia , Humanos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/biossíntese , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/imunologia , Testes Imunológicos/métodosRESUMO
The temperature-inducible λpL/pR-cI857 expression system has been widely used to produce recombinant proteins (RPs), especially when it is necessary to avoid the addition of exogenous materials to induce the expression of recombinant genes, preventing contamination of bioprocesses. The temperature increase favors the formation of inclusion bodies (IBs). The temperature upshift could change the metabolism, productivities, cell viability, IBs architecture, and the host cell proteins inside IBs, affecting downstream to obtain the final product. In this contribution, we focus on the relationship between the bioprocesses using temperature increase as inducer, the heat shock response associated with temperature up-shift, the RP accumulation, and the formation of IBs. Here, we describe how to produce IBs and how culture conditions can modulate the composition and architecture of IBs by modifying the induction temperature in RP production.
Assuntos
Escherichia coli , Corpos de Inclusão , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Corpos de Inclusão/metabolismo , Proteínas Recombinantes/biossíntese , TemperaturaRESUMO
Micrurus dumerilii is a coral snake of clinic interest in Colombia. Its venom is mainly composed of phospholipases A2 being MdumPLA2 the most abundant protein. Nevertheless, Micrurus species produce a low quantity of venom, which makes it difficult to produce anticoral antivenoms. Therefore, in this work, we present the recombinant expression of MdumPLA2 to evaluate its biological activities and its immunogenic potential to produce antivenoms. For this, a genetic construct rMdumPLA2 was cloned into the pET28a vector and expressed heterologously in bacteria. His-rMdumPLA2 was extracted from inclusion bodies, refolded in vitro, and isolated using affinity and RP-HPLC chromatography. His-rMdumPLA2 was shown to have phospholipase A2 activity, a weak anticoagulant effect, and induced myonecrosis and edema. The anti-His-rMdumPLA2 antibodies produced in rabbits recognized native PLA2, the complete venom of M. dumerilii, and a phospholipase from another species of the Micrurus genus. Antibodies neutralized 100% of the in vitro phospholipase activity of the recombinant toxin and a moderate percentage of the myotoxic activity of M. dumerilii venom in mice. These results indicate that His-rMdumPLA2 could be used as an immunogen to improve anticoral antivenoms development. This work is the first report of an M. dumerilii functional recombinant PLA2.
Assuntos
Antivenenos , Cobras Corais , Venenos Elapídicos , Fosfolipases A2 , Animais , Camundongos , Coelhos , Antivenenos/biossíntese , Antivenenos/genética , Antivenenos/imunologia , Venenos Elapídicos/enzimologia , Fosfolipases A2/biossíntese , Fosfolipases A2/genética , Fosfolipases A2/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
BACKGROUND: Native-like secondary structures and biological activity have been described for proteins in inclusion bodies (IBs). Tertiary structure analysis, however, is hampered due to the necessity of mild solubilization conditions. Denaturing reagents used for IBs solubilization generally lead to the loss of these structures and to consequent reaggregation due to intermolecular interactions among exposed hydrophobic domains after removal of the solubilization reagent. The use of mild, non-denaturing solubilization processes that maintain existing structures could allow tertiary structure analysis and increase the efficiency of refolding. RESULTS: In this study we use a variety of biophysical methods to analyze protein structure in human growth hormone IBs (hGH-IBs). hGH-IBs present native-like secondary and tertiary structures, as shown by far and near-UV CD analysis. hGH-IBs present similar λmax intrinsic Trp fluorescence to the native protein (334 nm), indicative of a native-like tertiary structure. Similar fluorescence behavior was also obtained for hGH solubilized from IBs and native hGH at pH 10.0 and 2.5 kbar and after decompression. hGH-IBs expressed in E. coli were extracted to high yield and purity (95%) and solubilized using non-denaturing conditions [2.4 kbar, 0.25 M arginine (pH 10), 10 mM DTT]. After decompression, the protein was incubated at pH 7.4 in the presence of the glutathione-oxidized glutathione (GSH-GSSG) pair which led to intramolecular disulfide bond formation and refolded hGH (81% yield). CONCLUSIONS: We have shown that hGH-IBs present native-like secondary and tertiary structures and that non-denaturing methods that aim to preserve them can lead to high yields of refolded protein. It is likely that the refolding process described can be extended to different proteins and may be particularly useful to reduce the pH required for alkaline solubilization.
Assuntos
Hormônio do Crescimento Humano , Corpos de Inclusão , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Hormônio do Crescimento Humano/metabolismo , Corpos de Inclusão/metabolismo , Redobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , SolubilidadeRESUMO
Epidermal growth factors (EGF) play a wide range of roles in embryogenesis, skin development, immune response homeostasis. They are involved in several pathologies as well, including several cancer types, psoriasis, chronic pain and chronic kidney disease. All members share the structural EGF domain, which is responsible for receptor interaction, thereby initiating transduction of signals. EGF growth factors have intense use in fundamental research and high potential for biotechnological applications. However, due to their structural organization with three disulfide bonds, recombinant production of these factors in prokaryotic systems is not straightforward. A significant fraction usually forms inclusion bodies. For the fraction remaining soluble, misfolding and incomplete disulfide bond formation may affect the amount of active factor in solution, which can compromise experimental conclusions and biotechnological applications. In this work, we describe a reliable procedure to produce seven human growth factors of the EGF family in Escherichia coli. Biophysical and stability analyses using limited proteolysis, light scattering, circular dichroism and nanoDSF show that the recombinant factors present folded and stable conformation. Cell proliferation and scratch healing assays confirmed that the recombinant factors are highly active at concentrations as low as 5 ng/ml.
Assuntos
Fator de Crescimento Epidérmico , Escherichia coli , Proliferação de Células , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Conformação Molecular , Proteínas Recombinantes/biossínteseRESUMO
BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.
Assuntos
Células CHO , Caspase 7/genética , Pontos de Checagem do Ciclo Celular , Proteínas Recombinantes/biossíntese , Animais , Divisão Celular , Cricetinae , Cricetulus , Técnicas de Inativação de Genes , Proteínas Recombinantes/genéticaRESUMO
The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low ß-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized ß-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The ß-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the ß-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, ß-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.
Assuntos
Enzimas Imobilizadas , Escherichia coli , Proteínas Fúngicas , Expressão Gênica , Hypocreales/genética , Nanopartículas de Magnetita/química , beta-Glucosidase , Estabilidade Enzimática , Enzimas Imobilizadas/biossíntese , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Hypocreales/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , beta-Glucosidase/biossíntese , beta-Glucosidase/química , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificaçãoRESUMO
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.
A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como ∆G 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.