RESUMEN
cGAS is a key cytosolic dsDNA receptor that senses viral infection and elicits interferon production through the cGAS-cGAMP-STING axis. cGAS is activated by dsDNA from viral and bacterial origins as well as dsDNA leaked from damaged mitochondria and nucleus. Eventually, cGAS activation launches the cell into an antiviral state to restrict the replication of both DNA and RNA viruses. Throughout the long co-evolution, viruses devise many strategies to evade cGAS detection or suppress cGAS activation. We recently reported that the Dengue virus protease NS2B3 proteolytically cleaves human cGAS in its N-terminal region, effectively reducing cGAS binding to DNA and consequent production of the second messenger cGAMP. Several other RNA viruses likely adopt the cleavage strategy. Here, we describe a protocol for the purification of recombinant human cGAS and Dengue NS2B3 protease, as well as the in vitro cleavage assay.
Asunto(s)
Virus del Dengue , Nucleotidiltransferasas , Proteínas no Estructurales Virales , Humanos , Proteínas no Estructurales Virales/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Proteolisis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Nucleótidos Cíclicos/metabolismo , Dengue/virología , Dengue/metabolismoRESUMEN
Commercial production of recombinant streptavidin (SAV) using soluble expression route is cost-prohibitive, resulting from its inherent toxicity toward commercially available Escherichia coli hosts (such as BL21) and low productivity of existing manufacturing processes. Quality challenges can also result from binding of streptavidin in the host cells. One way to overcome these challenges is to allow formation of inclusion bodies (IBs). Nevertheless, carried-over cellular contaminants during IBs preparation can hinder protein refolding and application of SAV in nucleic acid-based applications. Hence, removing associated contaminants in recombinant IBs is imperative for maximum product outcomes. In this study, the IBs isolation method from our group was improved to remove residual DNA found in refolded core SAV (cSAV). The improvements were attained by incorporating quantitative real-time polymerase chain reactions (qPCR) for residual DNA monitoring. We attained 99 % cellular DNA removal from cSAV IBs via additional wash and sonication steps, and the addition of benzonase nuclease during lysis. A 10 % increment of cSAV refolding yield (72 %) and 83 % reduction of residual DNA from refolding of 1 mg cSAV IBs were observed under extensive sonication. Refolding of cSAV was not affected and its activity was not compromised. The optimized process reported here highlights the importance of obtaining cSAV IBs with minimal contaminants prior to refolding to increase product yield, and the usefulness of the qPCR method to monitor nucleic acid removed from each step of the process.
Asunto(s)
Escherichia coli , Cuerpos de Inclusión , Replegamiento Proteico , Proteínas Recombinantes , Estreptavidina , Cuerpos de Inclusión/química , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Estreptavidina/química , Estreptavidina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesisRESUMEN
Signal peptide (SP) is required for secretion of recombinant proteins and typically cleaved by signal peptidase at its C-region to generate the mature proteins. Miscleavage of the SP is reported occasionally, resulting in a truncated- or elongated-terminal sequence. In the present work, we demonstrated that cation exchange (CEX) chromatography is an effective means for removing SP variants with a case study. With the selected resin/conditions, the chromatographic performance is comparable between runs performed at the low end and high end of load density and elution range. The procedure described in this work can be used as a general approach for resin selection and optimization of chromatographic conditions to remove byproducts that bind more strongly than the product to the selected resin.
Asunto(s)
Señales de Clasificación de Proteína , Cromatografía por Intercambio Iónico/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Resinas de Intercambio de Catión/química , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110α/p85α and a 3-fold increase in p110α. We also used these protocols to produce comparable constructs of the ß and δ isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I , Humanos , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Fosfatidilinositol 3-Quinasa Clase Ia/química , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/química , Solubilidad , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Essential amino acid, tryptophan which intake from food plays a critical role in numerous metabolic functions, exhibiting extensive biological functions and applications. Tryptophan is beneficial for the food sector by enhancing nutritional content and promoting the development of functional foods. A putative gene encoding tryptophan synthase was the first identified in Sphingobacterium soilsilvae Em02, a cellulosic bacterium making it inherently more environmentally friendly. The gene was cloned and expressed in exogenous host Escherichia coli, to elucidate its function. The recombinant tryptophan synthase with a molecular weight 42 KDa was expressed in soluble component. The enzymatic activity to tryptophan synthase in vivo was assessed using indole and L-serine and purified tryptophan synthase. The optimum enzymatic activity for tryptophan synthase was recorded at 50 ºC and pH 7.0, which was improved in the presence of metal ions Mg2+, Sr2+ and Mn2+, whereas Cu2+, Zn2+ and Co2+ proved to be inhibitory. Using site-directed mutagenesis, the consensus pattern HK-S-[GGGSN]-E-S in the tryptophan synthase was demonstrated with K100Q, S202A, G246A, E361A and S385A as the active sites. Tryptophan synthase has been demonstrated to possess the defining characteristics of the ß-subunits. The tryptophan synthase may eventually be useful for tryptophan production on a larger scale. Its diverse applications highlight the potential for improving both the quality and health benefits of food products, making it an essential component in advancing food science and technology.
Asunto(s)
Escherichia coli , Mutagénesis Sitio-Dirigida , Triptófano Sintasa , Triptófano , Triptófano Sintasa/metabolismo , Triptófano Sintasa/genética , Triptófano Sintasa/química , Triptófano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Sphingomonadaceae/enzimología , Sphingomonadaceae/genética , Sphingomonadaceae/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Dominio Catalítico , Clonación Molecular , Concentración de Iones de Hidrógeno , Indoles/metabolismo , Catálisis , Serina/metabolismoRESUMEN
The lipoxygenase cascade in plants is a source of oxylipins (oxidized fatty acid derivatives), which play an important role in regulatory processes and formation of plant response to stress factors. Some of the most common enzymes of the lipoxygenase cascade are 13-specific hydroperoxide lyases (HPLs, also called hemiacetal synthases) of the CYP74B subfamily. In this work, we identified and cloned the CYP74B34 gene from carrot (Daucus carota L.) and described the biochemical properties of the corresponding recombinant enzyme. The CYP74B34 enzyme was active towards 9- and 13-hydroperoxides of linoleic (9-HPOD and 13-HPOD, respectively) and α-linolenic (9-HPOT and 13-HPOT, respectively) acids. CYP74B34 specifically converted 9-HPOT and 13-HPOT into aldo acids (HPL products). The transformation of 13-HPOD led to the formation of aldo acids and epoxyalcohols [products of epoxyalcohol synthase (EAS) activity] as major and minor products, respectively. At the same time, conversion of 9-HPOD resulted in the formation of epoxyalcohols as the main products and aldo acids as the minor ones. Therefore, CYP74B34 is the first enzyme with a double HPL/EAS activity described in carrot. The presence of these catalytic activities was confirmed by analysis of the oxylipin profiles for the roots from young seedlings and mature plants. In addition, we substituted amino acid residues in one of the catalytically essential sites of the CYP74B34 and CYP74B33 proteins and investigated the properties of the obtained mutant enzymes.
Asunto(s)
Aldehído-Liasas , Sistema Enzimático del Citocromo P-450 , Daucus carota , Proteínas de Plantas , Daucus carota/enzimología , Daucus carota/genética , Daucus carota/metabolismo , Aldehído-Liasas/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Peróxidos Lipídicos/metabolismo , Especificidad por Sustrato , Secuencia de Aminoácidos , Ácidos LinoleicosRESUMEN
The cold-shock expression system in Escherichia coli was developed on a manual induction approach using optical density at 600 nm (OD600) measurements and isopropyl ß-D-1-thiogalactopyranoside (IPTG) addition. In this study, we show that cold-shock expression performs equally well using an autoinduction approach wherein OD600 measurements and IPTG addition may be eliminated. We further demonstrate that cold-shock expression with autoinduction can better facilitate high-throughput experiments.
Asunto(s)
Escherichia coli , Isopropil Tiogalactósido , Escherichia coli/genética , Escherichia coli/metabolismo , Isopropil Tiogalactósido/farmacología , Regulación Bacteriana de la Expresión Génica , Respuesta al Choque por Frío/genética , Frío , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
At present, biopharmaceuticals have received extensive attention from the society, among which recombinant proteins have a good growth trend and a large market share. Chinese hamster ovary (CHO) cells are the preferred mammalian system to produce glycosylated recombinant protein drugs. A highly efficient and stable cell screening method needs to be developed to obtain more and useful recombinant proteins. Limited dilution method, cell sorting, and semi-solid medium screening are currently the commonly used cell cloning methods. These methods are time-consuming and labor-intensive, and they have the disadvantage of low clone survival rate. Here, a method based on semi-solid medium was developed to screen out high-yielding and stable cell line within 3 weeks to improve the screening efficiency. The semi-solid medium was combined with an expression vector containing red fluorescent protein (RFP) for early cell line development. In accordance with the fluorescence intensity of RFP, the expression of upstream target gene could be indicated, and the fluorescence intensity was in direct proportion to the expression of upstream target gene. In conclusion, semi-solid medium combined with bicistronic expression vector provides an efficient method for screening stable and highly expressed cell lines.
Asunto(s)
Cricetulus , Proteínas Recombinantes , Células CHO , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Vectores Genéticos/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Cricetinae , Proteína Fluorescente Roja , Medios de Cultivo/químicaRESUMEN
The inner capsid protein of rotavirus, VP6, emerges as a promising candidate for next-generation vaccines against rotaviruses owing to its abundance in virion particles and high conservation. However, the formation of inclusion bodies during prokaryotic VP6 expression poses a significant hurdle to rotavirus research and applications. Here, we employed experimental and computational approaches to investigate inclusion body formation and aggregation-prone regions (APRs). Heterologous recombinant VP6 expression in Escherichia coli BL21(DE3) cells resulted in inclusion body formation, confirmed by transmission electron microscopy revealing amorphous aggregates. Thioflavin T assay demonstrated incubation temperature-dependent aggregation of VP6 inclusion bodies. Computational predictions of APRs in rotavirus A VP6 protein were performed using sequence-based tools (TANGO, AGGRESCAN, Zyggregator, Waltz, FoldAmyloid, ANuPP, Camsol intrinsic) and structure-based tools (SolubiS, CamSol structurally corrected, Aggrescan3D). A total of 24 consensus APRs were identified, with 21 of them being surface-exposed in VP6. All identified APRs display a predominance of hydrophobic amino acids, ranging from 33 to 100%. Computational identification of these APRs corroborates our experimental observation of VP6 inclusion body or aggregate formation. Characterization of VP6's aggregation propensity facilitates understanding of its behaviour during prokaryotic expression and opens avenues for protein engineering of soluble variants, advancing research on rotavirus VP6 in pathology, therapy, and diagnostics.
Asunto(s)
Antígenos Virales , Proteínas de la Cápside , Escherichia coli , Cuerpos de Inclusión , Rotavirus , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Antígenos Virales/genética , Antígenos Virales/metabolismo , Cuerpos de Inclusión/metabolismo , Rotavirus/genética , Rotavirus/metabolismo , Agregado de Proteínas , Simulación por Computador , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Corynebacterium glutamicum is an attractive host for secretory production of recombinant proteins, including high-value industrial enzymes and therapeutic proteins. The choice of an appropriate signaling peptide is crucial for efficient protein secretion. However, due to the limited availability of signal peptides in C. glutamicum, establishing an optimal secretion system is challenging. RESULT: We constructed a signal peptide library for the isolation of target-specific signal peptides and developed a highly efficient secretory production system in C. glutamicum. Based on the sequence information of the signal peptides of the general secretion-dependent pathway in C. glutamicum, a synthetic signal peptide library was designed, and validated with three protein models. First, we examined endoxylanase (XynA) and one potential signal peptide (C1) was successfully isolated by library screening on xylan-containing agar plates. With this C1 signal peptide, secretory production of XynA as high as 3.2 g/L could be achieved with high purity (> 80%). Next, the signal peptide for âº-amylase (AmyA) was screened on a starch-containing agar plate. The production titer of the isolated signal peptide (HS06) reached 1.48 g/L which was 2-fold higher than that of the well-known Cg1514 signal peptide. Finally, we isolated the signal peptide for the M18 single-chain variable fragment (scFv). As an enzyme-independent screening tool, we developed a fluorescence-dependent screening tool using Fluorescence-Activating and Absorption-Shifting Tag (FAST) fusion, and successfully isolated the optimal signal peptide (18F11) for M18 scFv. With 18F11, secretory production as high as 228 mg/L was achieved, which was 3.4-fold higher than previous results. CONCLUSIONS: By screening a fully synthetic signal peptide library, we achieved improved production of target proteins compared to previous results using well-known signal peptides. Our synthetic library provides a useful resource for the development of an optimal secretion system for various recombinant proteins in C. glutamicum, and we believe this bacterium to be a more promising workhorse for the bioindustry.
Asunto(s)
Corynebacterium glutamicum , Señales de Clasificación de Proteína , Proteínas Recombinantes , Corynebacterium glutamicum/metabolismo , Corynebacterium glutamicum/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Biblioteca de Péptidos , Endo-1,4-beta Xilanasas/metabolismo , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/biosíntesis , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , alfa-Amilasas/metabolismo , alfa-Amilasas/genéticaRESUMEN
The Trichinella spiralis novel cystatin (TsCstN) inhibits cathepsin L (CatL) activity and inflammation of macrophages during lipopolysaccharide (LPS) induction. To identify the protease inhibitory region, this study applied an in silico modeling approach to simulate truncation sites of TsCstN (Ts01), which created four truncated forms, including TsCstN∆1-39 (Ts02), TsCstN∆1-71 (Ts03), TsCstN∆1-20, ∆73-117 (Ts04), and TsCstN∆1-20, ∆42-117 (Ts05). The superimposition of these truncates modeled with AlphaFold Colab indicated that their structures were more akin to Ts01 than those modeled with I-TASSER. Moreover, Ts04 exhibited the closest resemblance to the structure of Ts01. The recombinant Ts01 (rTs01) and truncated proteins (rTs02, rTs03, and rTs04) were successfully expressed in a prokaryotic expression system while Ts05 was synthesized, with sizes of approximately 14, 12, 8, 10, and 2.5 kDa, respectively. When determining the inhibition of CatL activity, both rTs01 and rTs04 effectively reduced CatL activity in vitro. Thus, the combination of the α1 and L1 regions may be sufficient to inhibit CatL. This study provides comprehensive insights into TsCstN, particularly regarding its protein function and inhibitory domains against CatL.
Asunto(s)
Cistatinas , Trichinella spiralis , Trichinella spiralis/genética , Trichinella spiralis/metabolismo , Animales , Cistatinas/metabolismo , Cistatinas/química , Cistatinas/genética , Catepsina L/metabolismo , Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Proteínas del Helminto/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Modelos Moleculares , Dominios Proteicos , Ratones , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Lipopolisacáridos/farmacologíaRESUMEN
Pathogenic spirochetes of the genus Leptospira are the causative agent of leptospirosis, a widely disseminated zoonosis that affects humans and animals. The ability of leptospires to quickly cross host barriers causing infection is not yet fully understood. Thus, understanding the mechanisms of pathogenicity is important to combat leptospiral infection. Outer membrane proteins are interesting targets to study as they are able to interact with host molecules. Proteins containing leucine-rich repeat (LRR) domains are characterized by the presence of multiple regions containing leucine residues and they have putative functions related to host-pathogen interactions. Hence, the present study aimed to clone and express the recombinant protein encoded by the LIC11098 gene, an LRR protein of L. interrogans serovar Copenhageni. In silico analyses predicted that the target protein is conserved among pathogenic strains of Leptospira, having a signal peptide and multiple LRR domains. The DNA sequence encoding the LRR protein was cloned in frame into the pAE vector, expressed without mutations in Escherichia coli and purified by His-tag chromatography. Circular dichroism (CD) spectrum showed that the recombinant protein was predominantly composed of ß-sheets. A dose-dependent interaction was observed with cellular and plasma fibronectins, laminin and the complement system component C9, suggesting a possible role of the protein encoded by LIC11098 gene at the initial stages of infection.
Asunto(s)
Leptospira interrogans , Proteínas Repetidas Ricas en Leucina , Proteínas Recombinantes , Leptospira interrogans/genética , Leptospira interrogans/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Simulación por Computador , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Clonación Molecular , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Leptospirosis/microbiología , Animales , Interacciones Huésped-Patógeno , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Dicroismo Circular , Secuencia de AminoácidosRESUMEN
The energetic metabolism of cancer cells relies on a substantial commitment of pyruvate to the catalytic action of lactate-generating dehydrogenases. This coupling mainly depends on lactate dehydrogenase A (LDH-A), which is overexpressed in different types of cancers, and therefore represents an appealing therapeutic target. Taking into account that the activity of LDHs is exclusively exerted by their tetrameric forms, it was recently shown that peptides perturbing the monomers-to-tetramer assembly inhibit human LDH-A (hLDH-A). However, to identify these peptides, tetrameric hLDH-A was transiently exposed to strongly acidic conditions inducing its dissociation into monomers, which were tested as a target for peptides at low pH. Nevertheless, the availability of native monomeric hLDH-A would allow performing similar screenings under physiological conditions. Here we report on the unprecedented isolation of recombinant monomeric hLDH-A at neutral pH, and on its use to identify peptides inhibiting the assembly of the tetrameric enzyme. Remarkably, the GQNGISDL octapeptide, mimicking the 296-303 portion of hLDH-A C-terminal region, was observed to effectively inhibit the target enzyme. Moreover, by dissecting the action of this octapeptide, the cGQND cyclic tetrapeptide was found to act as the parental compound. Furthermore, we performed assays using MCF7 and BxPC3 cultured cells, exclusively expressing hLDH-A and hLDH-B, respectively. By means of these assays we detected a selective action of linear and cyclic GQND tetrapeptides, inhibiting lactate secretion in MCF7 cells only. Overall, our observations suggest that peptides mimicking the C-terminal region of hLDH-A effectively interfere with protein-protein interactions responsible for the assembly of the tetrameric enzyme.
Asunto(s)
L-Lactato Deshidrogenasa , Ácido Láctico , Multimerización de Proteína , Humanos , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/química , Ácido Láctico/metabolismo , Ácido Láctico/química , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Concentración de Iones de Hidrógeno , Oligopéptidos/química , Oligopéptidos/metabolismo , Oligopéptidos/genética , Oligopéptidos/farmacología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Línea Celular TumoralRESUMEN
Transcriptional regulatory networks (TRNs) associated with recombinant protein (rProt) synthesis in Yarrowia lipolytica are still under-described. Yet, it is foreseen that skillful manipulation with TRNs would enable global fine-tuning of the host strain's metabolism towards a high-level-producing phenotype. Our previous studies investigated the transcriptomes of Y. lipolytica strains overproducing biochemically different rProts and the functional impact of transcription factors (TFs) overexpression (OE) on rProt synthesis capacity in this species. Hence, much knowledge has been accumulated and deposited in public repositories. In this study, we combined both biological datasets and enriched them with further experimental data to investigate an interplay between TFs and rProts synthesis in Y. lipolytica at transcriptional and functional levels. Technically, the RNAseq datasets were extracted and re-analyzed for the TFs' expression profiles. Of the 140 TFs in Y. lipolytica, 87 TF-encoding genes were significantly deregulated in at least one of the strains. The expression profiles were juxtaposed against the rProt amounts from 125 strains co-overexpressing TF and rProt. In addition, several strains bearing knock-outs (KOs) in the TF loci were analyzed to get more insight into their actual involvement in rProt synthesis. Different profiles of the TFs' transcriptional deregulation and the impact of their OE or KO on rProts synthesis were observed, and new engineering targets were pointed.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas Recombinantes , Factores de Transcripción , Yarrowia , Yarrowia/genética , Yarrowia/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Redes Reguladoras de Genes , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Transcriptoma , Perfilación de la Expresión Génica , Transcripción GenéticaRESUMEN
l-Homocysteine, formed from S-adenosyl methionine following demethylation and adenosine release, accumulates when the methionine recycling pathway and other pathways become impaired, thus leading to hyperhomocysteinemia, a biomarker in cardiovascular diseases, neurological/psychiatric disorders, and cancer. The partial oxidation of the l-homocysteine thiol group and its decarboxylation on C-alpha lead to the formation of l-homocysteinesulfinic acid (l-HCSA) and homohypotaurine (HHT), respectively. Both compounds are not readily available from commercial suppliers, which hinders the investigation of their biological activities. Herein, the chemical synthesis of l-HCSA, from l-homocystine, was the starting point for establishing the bio-based synthesis of HHT using recombinant Escherichia coli glutamate decarboxylase (EcGadB), an enzyme already successfully employed for the bio-based synthesis of GABA and its phosphinic analog. Prior to HHT synthesis, kcat (33.92 ± 1.07) and KM (38.24 ± 3.45 mM) kinetic constants were determined for l-HCSA on EcGadB. The results of our study show that the EcGadB-mediated synthesis of HHT can be achieved with good yields (i.e., 40% following enzymatic synthesis and column chromatography). Purified HHT was tested in vitro on primary human umbilical vein endothelial cells and rat cardiomyoblasts and compared to the fully oxidized analog, homotaurine (OT, also known as tramiprosate), in widespread pharmaceutical use. The results show that both cell lines display statistically significant recovery from the cytotoxic effects induced by H2O2 in the presence of HHT.
Asunto(s)
Escherichia coli , Glutamato Descarboxilasa , Homocisteína , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Humanos , Homocisteína/análogos & derivados , Homocisteína/metabolismo , Glutamato Descarboxilasa/metabolismo , Glutamato Descarboxilasa/genética , Proteínas Recombinantes/metabolismo , CinéticaRESUMEN
Background: Transmission-blocking vaccines (TBVs) can effectively prevent the community's spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in P. vivax. Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in P. vivax. Methods and findings: The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax isolates (named isolates #1-4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The anti-rPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the P.vivax Salvador (Sal) I strain sequences, the pvqsox in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution. Conclusions: Our results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization.
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Anticuerpos Antiprotozoarios , Vacunas contra la Malaria , Malaria Vivax , Plasmodium berghei , Plasmodium vivax , Proteínas Recombinantes , Plasmodium vivax/inmunología , Plasmodium vivax/genética , Plasmodium vivax/enzimología , Animales , Conejos , Vacunas contra la Malaria/inmunología , Anticuerpos Antiprotozoarios/inmunología , Malaria Vivax/prevención & control , Malaria Vivax/transmisión , Malaria Vivax/inmunología , Plasmodium berghei/inmunología , Plasmodium berghei/genética , Plasmodium berghei/enzimología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ratones , Escherichia coli/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Femenino , Humanos , Inmunogenicidad Vacunal , Ratones Endogámicos BALB CRESUMEN
Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O2) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components.
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Oxidación-Reducción , Oxigenasas , Oxigenasas/metabolismo , Oxigenasas/química , Oxigenasas/genética , Oxigenasas/aislamiento & purificación , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Rhodococcus/enzimología , Rhodococcus/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/químicaRESUMEN
Background: Toxoplasma gondii (T. gondii) is a widespread, zoonotic protozoan intracellular parasite with a complex life cycle, which can cause toxoplasmosis, a potentially serious disease. During the invasion process, T. gondii proteins first bind to the relevant host cell receptors, such as glycosaminoglycan molecule (GAG-binding motif), which is one of the main receptors for parasites or virus to infect host cells. However, research on TGME49_216510 (T. gondii Trx21), a protein from Toxoplasma gondii, is limited. Methods: Bioinformatics analysis of the Trx21 protein was performed firstly. And specific primers were then designed using the conserved domain and GAG-binding motif to amplify, express, and purify a fragment of the Trx21 protein. The purified Trx21-GST protein was used for antioxidant and cell adhesion experiments. Simultaneously, mice were immunized with Trx21-His to generate specific polyclonal antibodies for subcellular localization analysis. Results: The Trx21 protein, consisting of 774 amino acids, included a transmembrane region, three GAG-binding motifs, and a Thioredoxin-like domain. The recombinant Trx21-His protein had a molecular mass of about 31 kDa, while the Trx21-GST protein had a molecular mass of about 55 kDa, which was analyzed by SDS-PAGE and Western blot. Subcellular localization analysis by IFA revealed that Trx21 is predominantly distributed in the cytoplasm of T. gondii. Furthermore, Trx21 exhibited a protective effect on supercoiled DNA against metal-catalyzed oxidation (MCO) and demonstrated adhesion abilities to Vero cells. Conclusions: These results indicate that Trx21 plays an important role in host cell interaction and oxidative damage.
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Adhesión Celular , Proteínas Protozoarias , Tiorredoxinas , Toxoplasma , Toxoplasma/metabolismo , Toxoplasma/genética , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Animales , Ratones , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Antioxidantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Biología Computacional , Células Vero , Chlorocebus aethiops , Toxoplasmosis/parasitología , HumanosRESUMEN
Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1-184) and a hydrophobic C-terminal domain (residues 185-243). When a recombinant fusion protein construct [bacterial pelB leader sequence - human apoA-I (1-243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1-184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1-184) was secreted from the bacteria. When the pelB-apoA-I (1-184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1-184) was the sole major protein present. Incubation of apoA-I (1-184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein.
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Apolipoproteína A-I , Escherichia coli , Proteínas Recombinantes de Fusión , Apolipoproteína A-I/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/química , Lipoproteínas HDL/genéticaRESUMEN
Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase involved in human brain development and functioning. Mutations in CDKL5, especially in its catalytic domain, cause a severe developmental condition named CDKL5 deficiency disorder. Nevertheless, molecular studies investigating the structural consequences of such mutations are still missing. The CDKL5 catalytic domain harbors different sites of post-translational modification, such as phosphorylations, but their role in catalytic activity, protein folding, and stability has not been entirely investigated. With this work, we describe the expression pattern of the CDKL5 catalytic domain in Escherichia coli demonstrating that it predominantly aggregates. However, the use of solubility tags, the lowering of the expression temperature, the manual codon optimization to overcome an internal translational start, and the incubation of the protein with K+ and MgATP allow the collection of a soluble catalytically active kinase. Interestingly, the resulting protein exhibits hypophosphorylation compared to its eukaryotic counterpart, proving that bacteria are a useful tool to achieve almost unmodified CDKL5. Posing questions about the CDKL5 autoactivation mechanism and the determinants for its stability, this research provides a valuable platform for comparative biophysical studies between bacterial and eukaryotic-expressed proteins, contributing to our understanding of neurodevelopmental disorders associated with CDKL5 dysfunction.