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(1) Background: Producing active antimicrobial peptides with disulfide bonds in bacterial strains is challenging. The cytoplasm of Escherichia coli has a reducing environment, which is not favorable to the formation of disulfide bonds. Additionally, E. coli may express proteins as insoluble aggregates known as inclusion bodies and have proteolytic systems that can degrade recombinant peptides. Using E. coli strains like SHuffle and tagging the peptides with fusion proteins is a common strategy to overcome these difficulties. Still, the larger size of carrier proteins can affect the final yield of recombinant peptides. Therefore, a small fusion protein that can be purified using affinity chromatography may be an ideal strategy for producing antimicrobial peptides in E. coli. (2) Methods: In this study, we investigated the use of the small metal-binding protein SmbP as a fusion partner for expressing and purifying the antimicrobial peptide scygonadin in E. coli. Two constructs were designed: a monomer and a tandem repeat; both were tagged with SmbP at the N-terminus. The constructs were expressed in E. coli SHuffle T7 and purified using immobilized metal-affinity chromatography. Finally, their antimicrobial activity was determined against Staphylococcus aureus. (3) Results: SmbP is a remarkable fusion partner for purifying both scygonadin constructs, yielding around 20 mg for the monomer and 30 mg for the tandem repeat per 1 mL of IMAC column, reaching 95% purity. Both protein constructs demonstrated antimicrobial activity against S. aureus at MICs of 4 µM and 40 µM, respectively. (4) Conclusions: This study demonstrates the potential of SmbP for producing active peptides for therapeutic applications. The two scygonadin constructs in this work showed promising antimicrobial activity against S. aureus, suggesting they could be potential candidates for developing new antimicrobial drugs.

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Introduction: The S-layer proteins are a class of self-assembling proteins that form bi-dimensional lattices named S-Layer on the cell surface of bacteria and archaea. The protein SlpA, which is the major constituent of the Lactobacillus acidophilus S-layer, contains in its C-terminus region (SlpA284 - 444), a protein domain (named here as SLAPTAG) responsible for the association of SlpA to the bacterial surface. SLAPTAG was adapted for the development of a novel affinity chromatography method: the SLAPTAG-based affinity chromatography (SAC). Methods: Proteins with different molecular weights or biochemical functions were fused in-frame to the SLAPTAG and efficiently purified by a Bacillus subtilis-derived affinity matrix (named Bio-Matrix or BM). Different binding and elution conditions were evaluated to establish an optimized protocol. Results: The binding equilibrium between SLAPTAG and BM was reached after a few minutes of incubation at 4°C, with an apparent dissociation constant (KD) of 4.3µM. A reporter protein (H6-GFP-SLAPTAG) was used to compare SAC protein purification efficiency against commercial immobilized metal affinity chromatography. No differences in protein purification performance were observed between the two methods. The stability and reusability of the BM were evaluated, and it was found that the matrix remained stable for more than a year. BM could be reused up to five times without a significant loss in performance. Additionally, the recovery of bound SLAP-tagged proteins was explored using proteolysis with a SLAP-tagged version of the HRV-3c protease (SLAPASE). This released the untagged GFP while the cut SLAPTAG and the SLAPASE were retained in the BM. As an alternative, iron nanoparticles were linked to the BM, resulting in BMmag. The BMmag was successfully adapted for a magnetic SAC, a technique with potential applications in high-throughput protein production and purification. Discussion: The SAC protocol can be adapted as a universal tool for the purification of recombinant proteins. Furthermore, the SAC protocol utilizes simple and low-cost reagents, making it suitable for in-house protein purification systems in laboratories worldwide. This enables the production of pure recombinant proteins for research, diagnosis, and the food industry.

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The ε4 allele of the apolipoprotein E gene (APOE4) constitutes the main genetic risk factor for late-onset Alzheimer disease (AD). High amounts of pure apolipoprotein E4 (ApoE4), in a rapid and reproducible fashion, could be of value for studying its pathophysiological roles in AD. The aim of the present work was to optimize a preparative method to obtain highly purified recombinant ApoE4 (rApoE4) with full biological activity. rApoE4 was expressed in the E. Coli BL21(D3) strain and a soluble form of the protein was purified by a combination of affinity and size-exclusion chromatography that precluded a denaturation step. The structural integrity and the biochemical activity of the purified rApoE4 were confirmed by circular dichroism and a lipid-binding assay. Several biological parameters affected by rApoE4, such as mitochondrial morphology, mitochondrial membrane potential and reactive oxygen species production were studied in CNh cells, a neuronal cell line, and neurodifferentiation and dendritogenesis were analyzed in the SH-SY5Y neuroblastoma cell line. The improved rApoE4 purification technique reported here enables the production of highly purified protein that retain the structural properties and functional activity of the native protein, as confirmed by tests in two different neuronal cell lines in culture.

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This study proposed the development of a monolithic supermacroporous affinity column for direct capture of lactoperoxidase, a glycoprotein present in milk, whey, and colostrum, with several applications due to its wide antimicrobial activity. A poly(acrylamide)-based cryogel was produced by radical co-polymerization of monomers in frozen aqueous solution and activated with p-aminobenzenesulfonamide as a ligand for specific interaction with the lactoperoxidase. The axial liquid dispersion coefficients at different liquid flow rates were determined by measuring residence time distributions using the tracer pulse-response method. The axial dispersion coefficient was low and the height equivalent to theoretical plate was not dependent on the flow velocity. The adsorptive capacity of affinity cryogel was studied as a function of flow velocity and the best condition was 0.9 cm/min. The response surface methodology was applied to optimize the capture of the enzyme, as a function of pH and salt concentration. Higher purification factor value was found at a salt concentration of 80 mmol/L and pH of 8.0 (p < 0.05). There was no influence of the variables under study on the yield (p > 0.05). The results indicated that affinity cryogel is a promising chromatography support for the use in high-throughput one-step purification of lactoperoxidase from whey.

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Recombinant proteins have revolutionized modern medicine and therapeutics. Currently recombinant proteins are used to treat diseases of great worldwide impact, as they allow maintenance and improvement of the life’s quality of patients with rare genetic diseases, metabolic syndromes, and immunological diseases. Since 1982, after the production of the first therapeutic recombinant protein (PRT), recombinant insulin, all major pharmaceutical companies have tried to develop PRT and supported its growth in the market. The right choice of expression systems and purification processes become essential steps to produce high-quality recombinant proteins with high yield. The recombinant protein Lsa63, the target of this project, is a leptospiral adhesin involved in the bacteria adhesion to the host. Lsa63 is expressed on the surface of patogenic Leptospiras and is absent in saprophytic ones. That said, the use of this protein in the development of new therapies and diagnosis for leptospirosis could be interesting. During this project, Lsa63 was purified from the frozen biomass of E. coli previously produced using liquid chromatography techniques). In all purification steps Lsa63 was quantified by BCA (bicinchoninic acid) and analyzed by SDS-PAGE electrophoresis and Western Blotting. The relative purity of Lsa63 was estimated by densitometry of SDS-PAGE bands. Immobilized-metal affinity chromatography (IMAC) was chosen as the first purification step and 24.4% of Lsa63 was yield with relative purity of 54.8%, followed by size-exclusion chromatography (SEC) with recovery of 95.2% and last step ion-exchange chromatography (IEC) with 7.9% and 80.3% of relative purity. Despite having obtained 80.3% of relative purity the global yield was low (7.9%).

As proteínas recombinantes revolucionaram a medicina moderna e a terapêutica. Por isso, atualmente, as proteínas recombinantes são utilizadas para tratamento de doenças de grande impacto mundial, pois permitem manutenção e melhoria da qualidade de vida de pacientes portadores de doenças genéticas raras, síndromes metabólicas e doenças imunológicas. Desde 1982, após a produção da primeira proteína recombinante terapêutica (PRT), insulina recombinante, todas as grandes farmacêuticas começaram a desenvolver PRT e impulsionaram o crescimento destas no mercado. A escolha correta dos sistemas de expressão e processos de purificação tornam-se etapas essenciais para produção de proteínas recombinantes de alta qualidade e em alto rendimento. A proteína recombinante Lsa63, alvo deste projeto, é uma adesina de leptospira envolvida na adesão da bactéria no organismo dos hospedeiros. A Lsa63 é expressa na superfície de Leptospiras patogênicas e apresenta-se ausente em cepas saprofíticas. Dito isto, a utilização desta proteína, no desenvolvimento de novas terapias e diagnóstico para leptospirose pode ser interessante. Durante o decorrer deste projeto, a Lsa63 foi purificada a partir da biomassa congelada de E. coli utilizando técnicas de cromatografia líquida. Em todas as etapas de purificação a Lsa63 foi quantificada por BCA (ácido bicinchonínico) e analisada por eletroforese SDS-PAGE e Western Blotting. A pureza relativa da Lsa63 foi estimada por densitometria das bandas do gel de eletroforese. A cromatografia de afinidade por íons metálicos imobilizados (IMAC) foi escolhida como primeira etapa de purificação sendo que a Lsa63 foi obtida com rendimento de 24,4% e pureza relativa de 54,8%, seguida pela cromatografia de exclusão molecular com recuperação de 95,2% e a última etapa a cromatografia de troca iônica com recuperação de 52,0% e pureza relativa de 80,3%. Apesar de ter obtido uma pureza relativa de 80,3% a recuperação global incluindo todas as etapas cromatográficas foi baixa sendo 7,9%.

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U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.

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Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant Streptococcus pyogenes Cas9 (SpCas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (SgRNAs) or for the DNA-free editing of genes of interest. Hereby, we provide a simple and detailed expression and purification protocol for SpCas9 as a protein fused to GFP and MBP. This protocol improves protein yield and simplifies the purification process by overcoming the frequently occurring obstacles such as plasmid loss, inconsistent protein expression levels, or inadequate protein binding to affinity resins. On average, this protocol yields 10 to 30 mg of purified, active, His6−MBP−SpCas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%.

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The COVID-19 pandemic has demanded a range of biotechnological products for detection of SARS-CoV-2 variants and evaluation of human seroconversion after infection or vaccination. In this work, we describe an easy pipeline for expression of SARS-CoV-2 nucleocapsid (N) protein in insect cells followed by its purification via affinity chromatography. The N gene was cloned into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via transposition and the resulting recombinant baculovirus was used for infection of lepidopteran Sf9 cells adapted to high-density suspension. Using Tris-HCl pH 8.0 buffer as mobile phase and eluting bound proteins with 175 mM imidazole as part of a three-step gradient, an average of 1 mg N protein could be purified from each 50 mg of total protein from clarified supernatant. Such protein amount allows the manufacturing of serological tests and the development of basic studies on cellular responses to SARS-CoV-2.

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Annually, about 40,000 persons are stricken by snake bite accidents in Brazil, being most of the cases are inflicted by Bothrops genus, whose venom can induce serious pathophysiological effects, mainly disorders in hemostasis. Bothrops jararaca venom is characterized by three main actions: proteolytic, coagulant and hemorrhagic. Some clotting factors can be activated by components present in the venom, classified as pro-coagulant activators, responsible for activating factor X or factor II of the coagulation cascade, generating thrombin. So far, only one procoagulant activator of Bothrops jararaca venom BjV has been characterized, bothrojaractivase, a 23 kDa PI-type metalloproteinase. In this work, we used chromatographic techniques to isolate other procoagulant components of BjV. Thus, by means of affinity chromatographies, BjV was purified on HisTrap and Blue Sepharose resins. Fractions capable of coagulating human fibrinogen and rabbit plasma were obtained, and this last substrate is only coagulated by pro-coagulant activators of BjV. Such fractions with high clotting activity were analyzed, concentrated and their activities on different substrates were differentiated by the use of specific inhibitors of metalloproteinases and serine proteinases. Studies are still being carried out to understand which coagulation factors are being activated by these fractions, by using chromogenic substrates, electrophoresis runs, and mass spectrometry.

Anualmente, cerca de 40.000 pessoas são acometidas por acidentes ofídicos no Brasil, sendo a maioria dos casos decorrentes de serpentes do gênero Bothrops, cujo veneno pode induzir graves efeitos fisiopatológicos, principalmente distúrbios na hemostasia. O veneno da espécie Bothrops jararaca é caracterizado por três ações principais: proteolítica, coagulante e hemorrágica. Relacionado à atividade coagulante, alguns fatores da cascata de coagulação podem ser ativados por componentes presentes no veneno, classificados como ativadores do tipo pró-coagulantes, responsáveis por ativar o fator X ou o fator II da cascata, gerando trombina. Até o momento, apenas um ativador pró-coagulante do veneno de Bothrops jararaca BjV foi caracterizado, a bothrojaractivase, uma metaloproteinase do tipo PI, de 23 kDa. Neste trabalho, utilizamos técnicas cromatográficas para isolar outros componentes pró-coagulantes do BjV. Deste modo, por meio de cromatografias de afinidade, o BjV foi purificado em resinas de HisTrap e Blue Sepharose. Foram obtidas frações capazes de coagular o fibrinogênio humano e plasma de coelho, um substrato que somente é coagulado por enzimas pró-coagulantes do BjV. Tais frações com altas atividades coagulantes foram analisadas, concentradas e suas atividades sobre diferentes substratos foram diferenciadas pelo uso de inibidores específicos de metaloproteinases e serinaproteinases. Obtivemos duas frações parcialmente purificadas, com alta atividade específica, e que são inibidas por quelante de metais divalentes o-fenantrolina. Essas frações são capazes de coagular o plasma de coelho, sem coagular o fibrinogênio humano. Estudos ainda estão sendo realizados para entender quais fatores da cascata de coagulação estas frações ativam, pela utilização de substratos cromogênicos específicos, análises eletroforéticas e espectrometria de massa.

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Posttranslational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and interaction, and they are associated with a wide range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are often present in substoichiometric levels, and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task, requiring highly specialized and sensitive PTM-specific enrichment methods. Currently, several methods have been implemented for PTM enrichment, and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for the vast majority of more than 400 known modifications, we have no or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution are subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. Furthermore, the lysine-acetylated peptides present in the first TiO2 flow-through fraction are enriched by immunoprecipitation (IP) after peptide cleanup. Finally, the samples are fractionated by high pH reversed phase chromatography (HpH) or hydrophilic interaction liquid chromatography (HILIC ) to reduce sample complexity and increase the coverage in the subsequent LC-MS /MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.

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