RESUMO
Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function - dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles. Here we show that light-gated recruitment of a solubilizing domain, maltose-binding protein (MBP), results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP by disrupting condensation of the oncogenic fusion protein FUS-CHOP and reverting FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.
Assuntos
Condensados Biomoleculares , Luz , Proteínas Ligantes de Maltose , Optogenética , Proteína FUS de Ligação a RNA , Solubilidade , Proteínas Ligantes de Maltose/metabolismo , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Humanos , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Optogenética/métodos , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/química , Células HeLaRESUMO
Developing more effective bioactive ingredients of natural origin is imperative for promoting wound healing. Sea cucumbers have long enjoyed a good reputation as both food delicacies and traditional medicines. In this study, we heterogeneously expressed a Apostichopus japonicus derived novel protein AjPSPLP-3, which exhibits a theoretical molecular weight of 13.034 kDa, through fusion with maltose binding protein (MBP). AjPSPLP-3 contains a strict CXXCXC motif, nine extremely conserved cysteine residues and two highly conserved cysteine residues. The predicted structure of AjPSPLP-3 consists of random coil and nine ß-sheets, Cys30-Cys67, Cys38-Cys58, Cys53-Cys90, Cys56-Cys66, and Cys81-Cys102 participating in the formation of five pairs of disulfide bonds. In vitro experiments conducted on HaCaT cells proved that AjPSPLP-3 and MBP-fused AjPSPLP-3 significantly contribute to HaCaT cells proliferation and migration without exhibiting hemolytic activity on murine erythrocytes. Specifically, treatment with 10 µmol/L MBP-fused AjPSPLP-3 protein increased the viability of HaCaT cells by 12.28 % (p < 0.001), while treatment with 10 µmol/L AjPSPLP-3 protein increased viability of HaCaT cells by 6.01 % (p < 0.01). Furthermore, wound closure of MBP-fused AjPSPLP-3 and AjPSPLP-3 were 22.51 % (p < 0.01) and 7.32 % (p < 0.05) higher than that of the control groups in HaCaT cells following 24 h of incubation.
Assuntos
Movimento Celular , Proliferação de Células , Stichopus , Animais , Stichopus/genética , Stichopus/química , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Humanos , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Clonagem Molecular , Sequência de Aminoácidos , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismo , Células HaCaTRESUMO
Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.
Assuntos
Inteínas , Proteínas Ligantes de Maltose , Recombinases Rec A , beta-Galactosidase , Inteínas/genética , beta-Galactosidase/genética , beta-Galactosidase/química , beta-Galactosidase/isolamento & purificação , beta-Galactosidase/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/química , Recombinases Rec A/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Elastina/química , Elastina/genética , Elastina/isolamento & purificação , Precipitação Química , Escherichia coli/genética , Escherichia coli/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/químicaRESUMO
Bacterial toxins have received a great deal of attention in the development of cancer treatments. Parasporin-2 (PS2Aa1 or Mpp46Aa1) is a Bacillus thuringiensis parasporal protein that preferentially destroys human cancer cells while not harming normal cells, making it a promising anticancer treatment. With the efficient development and sustainable silver nanoparticles (AgNPs) synthesis technology, the biomedical use of AgNPs has expanded. This study presents the development of a novel nanotoxin composed of biosynthesized silver nanoparticles loaded with the N-terminal truncated PS2Aa1 toxin. MOEAgNPs were synthesized using a biological method, with Moringa oleifera leaf extract and maltose serving as reducing and capping agents. The phytochemicals present in M. oleifera leaf extract were identified by GC-MS analysis. MOEAgNPs were loaded with N-terminal truncated PS2Aa1 fused with maltose-binding protein (MBP-tPS2) to formulate PS2-MOEAgNPs. The PS2-MOEAgNPs were evaluated for size, stability, toxin loading efficacy, and cytotoxicity. PS2-MOEAgNPs demonstrated dose-dependent cytotoxicity against the T-cell leukemia MOLT-4 and Jurkat cell lines but had little effect on the Hs68 fibroblast or normal cell line. Altogether, the current study provides robust evidence that PS2-MOEAgNPs can efficiently inhibit the proliferation of T-cell leukemia cells, thereby suggesting their potential as an alternative to traditional anticancer treatments.
Assuntos
Antineoplásicos , Nanopartículas Metálicas , Prata , Humanos , Prata/química , Prata/farmacologia , Nanopartículas Metálicas/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Moringa oleifera/química , Proteínas Recombinantes/farmacologia , Folhas de Planta/química , Sobrevivência Celular/efeitos dos fármacos , Endotoxinas , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismoRESUMO
Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.
Assuntos
Escherichia coli , Interleucina-3 , Isomerases de Dissulfetos de Proteínas , Proteínas Recombinantes de Fusão , Humanos , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Interleucina-3/metabolismo , Interleucina-3/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Linhagem Celular Tumoral , SolubilidadeRESUMO
Cysteine conjugation is widely used to constrain phage displayed peptides for the selection of cyclic peptides against specific targets. In this study, the nontoxic Bi3+ ion was used as a cysteine conjugation reagent to cross-link peptide libraries without compromising phage infectivity. We constructed a randomized 3-cysteine peptide library and cyclized it with Bi3+, followed by a selection against the maltose-binding protein as a model target. Next-generation sequencing of selection samples revealed the enrichment of peptides containing clear consensus sequences. Chemically synthesized linear and Bi3+ cyclized peptides were used for affinity validation. The cyclized peptide showed a hundred-fold better affinity (0.31 ± 0.04 µM) than the linear form (39 ± 6 µM). Overall, our study proved the feasibility of developing Bi3+ constrained bicyclic peptides against a specific target using phage display, which would potentially accelerate the development of new peptide-bismuth bicycles for therapeutic or diagnostic applications.
Assuntos
Biblioteca de Peptídeos , Peptídeos Cíclicos , Peptídeos Cíclicos/química , Cisteína/química , Proteínas Ligantes de Maltose/metabolismo , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Ciclização , Peptídeos/química , Sequência de AminoácidosRESUMO
Polyphosphate exhibits a unique post-translational modification-like function, known as histidine polyphosphate modification (HPM), marked by a robust non-covalent interaction with histidine repeat proteins. Here, we present a protocol for detecting HPM of human proteins via maltose-binding protein-tagged expression in E. coli. We describe steps for detecting HPM by observing electrophoretic mobility shifts on NuPAGE gels followed by western blot. We then detail procedures for analyzing the influence of ionic strength and pH on HPM. For complete details on the use and execution of this protocol, please refer to Neville et al.1.
Assuntos
Escherichia coli , Histidina , Proteínas Ligantes de Maltose , Polifosfatos , Processamento de Proteína Pós-Traducional , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Polifosfatos/metabolismo , Polifosfatos/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Proteínas Ligantes de Maltose/química , Histidina/metabolismo , Histidina/genética , Histidina/química , Western BlottingRESUMO
Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in Escherichia coli often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar Kd) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications.
Assuntos
Escherichia coli , Anticorpos de Cadeia Única , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/química , Cromatografia de Afinidade , Proteínas Ligantes de Maltose/genéticaRESUMO
In protein biotechnology, large soluble fusion partners are widely utilized for increased yield and solubility of recombinant proteins. However, the production of additional large fusion partners poses an additional burden to the host, leading to a decreased protein yield. In this study, we identified two highly disordered short peptides that were able to increase the solubility of an artificially engineered aggregation-prone protein, GFP-GFIL4, from 0.6% to 61% (D3-DP00592) and 46% (D4-DP01038) selected from DisProt database. For further confirmation, the peptides were applied to two insoluble E. coli proteins (YagA and YdiU). The peptides also enhanced solubility from 52% to 90% (YagA) and from 27% to 93% (YdiU). Their ability to solubilize recombinant proteins was comparable with strong solubilizing tags, maltose-binding protein (40 kDa) and TrxA (12 kDa), but much smaller (< 7 kDa) in size. For practical application, the two peptides were fused with a restriction enzyme, I-SceI, and they increased I-SceI solubility from 24% up to 75%. The highly disordered peptides did not affect the activity of I-SceI while I-SceI fused with MBP or TrxA displayed no restriction activity. Despite the small size, the highly disordered peptides were able to solubilize recombinant proteins as efficiently as conventional fusion tags and did not interfere with the function of recombinant proteins. Consequently, the identified two highly disordered peptides would have practical utility in protein biotechnology and industry.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
There is a high demand for the production of recombinant proteins in Escherichia coli for biotechnological applications, but their production is still limited by their insolubility. Fusion tags have been successfully used to enhance the solubility of aggregation-prone proteins; however, smaller and more powerful tags are desired for increasing the yield and quality of target proteins. Here, the NEXT tag, a 53-amino-acid-long solubility enhancer, is described. The NEXT tag showed outstanding ability to improve both in vivo and in vitro solubilities, with minimal effect on passenger proteins. The C-terminal region of the tag was mostly responsible for in vitro solubility, while the N-terminal region was essential for in vivo soluble expression. The NEXT tag appeared to be intrinsically disordered and seemed to exclude neighboring molecules and prevent protein aggregation by acting as an entropic bristle. This novel peptide tag should have general use as a fusion partner to increase the yield and quality of difficult-to-express proteins. IMPORTANCE Production of recombinant proteins in Escherichia coli still suffers from the insolubility problem. Conventional solubility enhancers with large sizes, represented by maltose-binding protein (MBP), have remained the first-choice tags; however, the success of the soluble expression of tagged proteins is largely unpredictable. In addition, the large tags can negatively affect the function of target proteins. In this work, the NEXT tag, an intrinsically disordered peptide, was introduced as a small but powerful alternative to MBP. The NEXT tag could significantly improve both the expression level and the solubility of target proteins, including a thermostable carbonic anhydrase and a polyethylene terephthalate (PET)-degrading enzyme that are remarkable enzymes for environmental bioremediation.
Assuntos
Escherichia coli , Peptídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , SolubilidadeRESUMO
Complications related to atherosclerosis account for approximately 1 in 4 deaths in the United States and treatment has focused on lowering serum LDL-cholesterol levels with statins. However, approximately 50% of those diagnosed with atherosclerosis have blood cholesterol levels within normal parameters. Human fortilin is an anti-apoptotic protein and a factor in macrophage-mediated atherosclerosis and is hypothesized to protect inflammatory macrophages from apoptosis, leading to subsequent cardiac pathogenesis. Fortilin is unique because it provides a novel drug target for atherosclerosis that goes beyond lowering cholesterol and utilization of a solution nuclear magnetic resonance (NMR) spectroscopy, structure-based drug discovery approach requires milligram quantities of pure, bioactive, recombinant fortilin. Here, we designed expression constructs with different affinity tags and protease cleavage sites to find optimal conditions to obtain the quantity and purity of protein necessary for structure activity relationship studies. Plasmids encoding fortilin with maltose binding protein (MBP), 6-histidine (6His) and glutathione-S-transferase (GST), N- terminal affinity tags were expressed and purified from Escherichia coli (E. coli). Cleavage sites with tobacco etch virus (TEV) protease and human rhinovirus (HRV) 3C protease were assessed. Despite high levels of expression of soluble protein, the fusion constructs were resistant to proteinases without the inclusion of amino acids between the cleavage site and N-terminus. We surveyed constructs with increasing lengths of glycine/serine (GGS) linkers between the cleavage site and fortilin and found that inclusion of at least one GGS insert led to successful protease cleavage and pure fortilin with conserved binding to calcium as measured by NMR.
Assuntos
Cálcio/química , Proteínas Recombinantes de Fusão/genética , Proteína Tumoral 1 Controlada por Tradução/genética , Proteases Virais 3C/química , Sítios de Ligação , Cálcio/metabolismo , Clonagem Molecular , Endopeptidases/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Proteína Tumoral 1 Controlada por Tradução/química , Proteína Tumoral 1 Controlada por Tradução/metabolismoRESUMO
Fibroblast growth factor receptors (FGFRs) generate various transduction signals by interaction with fibroblast growth factors (FGFs) and are involved in various biological functions such as cell proliferation, migration, and differentiation. Malfunction of these proteins may lead to the development of various diseases, including cancer. Accordingly, FGFRs are considered an alternative therapeutic target for protein and/or gene therapy. However, the screening of antagonists or agonists of FGFRs is challenging due to their complex structural features associated with protein expression. Herein, we conducted the development of a protease-free cleavable tag (PFCT) for enhancing the solubility of difficult-to express protein by combining maltose-binding protein (MBP) and the C-terminal region of Npu intein. To validate the availability of the resulting tag for the functional production of extracellular domains of FGFRs (Ec_FGFRs), we performed fusion of PFCT with the N-terminus of Ec_FGFRs and analyzed the expression patterns. Almost all PFCT-Ec_FGFR fusion proteins were mainly detected in the soluble fraction except for Ec_FGFR4. Upon addition of the N-terminal region of Npu intein, approximately 85% of the PFCT-Ec_FGFRs was separated into PFCT and Ec_FGFR via intein-mediated cleavage. Additionally, the structural integrity of Ec_FGFR was confirmed by affinity purification using heparin column. Taken together, our study demonstrated that the PFCT could be used for soluble expression and selective separation of Ec_FGFRs.
Assuntos
Espaço Extracelular/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Humanos , Proteínas Ligantes de Maltose/genética , Fragmentos de Peptídeos/genética , Domínios Proteicos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.
Assuntos
ADP Ribose Transferases , Antineoplásicos Imunológicos/farmacologia , Toxinas Bacterianas , Exotoxinas , Imunotoxinas/farmacologia , Proteínas Ligantes de Maltose , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única , Fatores de Virulência , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Exotoxinas/genética , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Humanos , Imunotoxinas/genética , Imunotoxinas/isolamento & purificação , Proteínas Ligantes de Maltose/genética , Espectrometria de Massas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
The complete gag gene from small ruminant lentiviruses (SRLV) encodes for a polyprotein of 55 kDa, known as p55gag. p55gag presents multiple antigenic epitopes, which can be recognized by antibodies, increasing the opportunity to detect SRLV-positive animals. Therefore, this polyprotein is considered an excellent candidate to use in diagnostic tests to detect antibodies against SRLV. Different studies have suggested that the selection of the recombinant antigen, which must be representative of the virus strains circulating in the test population, is crucial to avoid false negative results. Thus, the use of proteins from different viral strains isolated from goats or sheep of a given region or country may be a useful strategy to increase the ability to detect SRLV-infected animals. In the present study, the pMAL-p5X vector was used to express and purify p55gag (now called rp55gag for recombinant polyprotein 55 gag). The cloned gene was inserted downstream from the malE gene of Escherichia coli, which encodes a maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein. The complete gag gene was amplified by RT-PCR. Finally, after digestion, the product was cloned into the pMAL-p5X vector and used to transform E. coli ER2325 cells. After the purification of MBP-rp55gag by affinity chromatography, the eluted fraction was observed by SDS-PAGE and Western Blot (WB). The WB was carried out with 85 serum samples from small ruminants previously analysed and compared by two commercial ELISAs. The results show that 76 of the serum samples were concordant with those by both ELISAs. Regarding the other nine serum samples, which showed discordant results between both ELISAs, were positive by WB. The results thus show that the rp55gag could be considered as an antigen in a confirmatory diagnostic assay to detect SRLV by WB. For this purpose, a future study with a high number of sera to determine the test specificity and sensitivity, using the p55gag of the circulating strain in Argentina will be necessary.
Assuntos
Doenças das Cabras , Infecções por Lentivirus , Doenças dos Ovinos , Animais , Escherichia coli , Doenças das Cabras/diagnóstico , Cabras , Lentivirus/genética , Infecções por Lentivirus/diagnóstico , Infecções por Lentivirus/veterinária , Proteínas Ligantes de Maltose/genética , Filogenia , Poliproteínas/genética , Ruminantes , Ovinos , Doenças dos Ovinos/diagnósticoRESUMO
The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due to their smaller size, low immunogenicity, and low-cost production. Although covalent strategies for the preparation of such ScFv-based therapeutic conjugates are prevalent, this approach is not straightforward, as it requires prior chemical activation and/or modification of both the ScFv and the therapeutics for the application of robust chemistries. A non-covalent alternative based on ScFv fused to maltose-binding protein (MBP) acting as a binding adapter is proposed for active targeted delivery. MBP-ScFv proves to be a valuable modular platform to synergistically bind maltose-derivatized therapeutic cargos through the MBP, while preserving the targeting competences provided by the ScFv. The methodology has been tested by using a mutated maltose-binding protein (MBP I334W) with an enhanced affinity toward maltose and an ScFv coding sequence toward the human epidermal growth factor receptor 2 (HER2). Non-covalent binding complexes of the resulting MBP-ScFv fusion protein with diverse maltosylated therapeutic cargos (a near-infrared dye, a maltosylated supramolecular ß-cyclodextrin container for doxorubicin, and non-viral polyplex gene vector) were easily prepared and characterized. In vitro and in vivo assays using cell lines that express or not the HER2 epitope, and mice xenografts of HER2 expressing cells demonstrated the capability and versatility of MBP-ScFv for diagnosis, imaging, and drug and plasmid active targeted tumor delivery. Remarkably, the modularity of the MBP-ScFv platform allows the flexible interchange of both the cargos and the coding sequence for the ScFv, allowing ad hoc solutions in targeting delivery without any further optimization since the MBP acts as a pivotal element.
Assuntos
Anticorpos de Cadeia Única , Animais , Anticorpos Monoclonais , Doxorrubicina , Maltose , Proteínas Ligantes de Maltose/genética , Camundongos , Anticorpos de Cadeia Única/genéticaRESUMO
The peptide hormone amylin receptor is a complex of the calcitonin receptor (CTR) and an accessory protein called receptor activity-modifying proteins (RAMPs). The soluble extracellular domain (ECD) of CTR is an important binding site of peptide hormone calcitonin. RAMPs also have an ECD and the association of CTR ECD with RAMP ECD enhances the affinity of peptide hormone amylin. However, the mechanism of how RAMP ECD association enhances amylin affinity remains elusive. Here, we report evidence supporting direct molecular interaction between an antagonistic amylin analog AC413 and RAMP2 ECD. We measured FITC-labeled peptide affinity for purified receptor ECD using fluorescence polarization (FP). We first found that RAMP2 ECD addition to maltose-binding protein (MBP)-tagged CTR ECD and an engineered MBP-tagged RAMP2 ECD-CTR ECD fusion protein (MBP-RAMP2-CTR ECD fusion) enhanced AC413 affinity. This suggests that these recombinant ECD systems represent functional amylin receptors. Interestingly, AC413 C-terminal residue Tyr25 (Y25) to Pro mutation eliminated its selective affinity for the MBP-RAMP2-CTR ECD fusion suggesting the critical role of the AC413 C-terminal residue in amylin receptor selectivity. Our structural model of the RAMP2 ECD:CTR ECD complex predicted molecular interaction of AC413 C-terminal residue Y25 with RAMP2 Glu101 (E101). Our FP peptide-binding assay showed that the RAMP2 E101A mutation of MBP-RAMP2-CTR ECD fusion decreased AC413 affinity by 7-fold, while the affinity of AC413 with the Y25P mutation was minimally changed. Consistently, AC413 binding affinity for the MBP-free RAMP2-CTR ECD fusion protein was also markedly decreased by the RAMP2 E101A mutation, while the affinity of AC413 with the Y25P mutation was moderately decreased. Together, our results support the molecular interaction between the AC413 C-terminal residue Y25 and RAMP2 E101 expanding our understanding of how the accessory protein RAMP2 enhances affinity of peptide hormone amylin for its receptor.
Assuntos
Polarização de Fluorescência , Peptídeos/química , Proteína 2 Modificadora da Atividade de Receptores/química , Animais , Humanos , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Modelos Moleculares , Peptídeos/genética , Ratos , Proteína 2 Modificadora da Atividade de Receptores/genética , Salmão , Alinhamento de SequênciaRESUMO
The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.
Assuntos
Cromatografia de Afinidade/métodos , Escherichia coli/genética , Histidina/genética , Oligopeptídeos/genética , Proteínas Recombinantes de Fusão/química , Coloração e Rotulagem/métodos , Epitopos/química , Epitopos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Histidina/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Oligopeptídeos/metabolismo , Peptídeo Hidrolases/química , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Domínios Proteicos , Proteólise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Solubilidade , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
The autocatalytic process of protein splicing is facilitated by an intein, which interrupts flanking polypeptides called exteins. The mechanism of protein splicing has been studied by overexpression in E. coli of intein fusion proteins with nonnative exteins. Inteins can be used to generate reactive α-thioesters, as well as proteins with N-terminal Cys residues, to facilitate expressed protein ligation. As such, a more detailed understanding of the function of inteins can have significant impact for biotechnology applications. Here, we provide biochemical methods to study splicing activity and NMR methods to study intein structure and the catalytic mechanism.
Assuntos
Biocatálise , Bioquímica/métodos , Inteínas , Processamento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Ácido Aspártico/química , Isótopos de Carbono , Cisteína/química , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Exteínas , Expressão Gênica , Vetores Genéticos/genética , Histidina/química , Concentração de Íons de Hidrogênio , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes de Fusão/biossínteseRESUMO
The Car9 affinity tag is a dodecameric silica-binding peptide that can be fused to the N- and C-termini of proteins of interest to enable their rapid and inexpensive purification on underivatized silica in a process that typically relies on l-lysine as an eluent. Here, we show that silica paper spin columns and borosilicate multi-well plates used for plasmid DNA purification are suitable for recovering Car9-tagged proteins with high purity in a workflow compatible with high-throughput experiments. Spin columns typically yield 100 µg of biologically active material that can be recovered in minutes with low concentrations of lysine. Because of their short bed length, spin columns also offer unique advantages, as evidenced by the selective recovery of functional Car9-tagged tobacco etch virus (TEV) protease from a fused and auto-cleaved maltose binding protein (MBP) folding partner that nonspecifically binds to silica in the presence of NaCl. These additional purification modalities should increase the versatility and appeal of the Car9 tag for affinity protein purification.
Assuntos
Cromatografia de Afinidade/métodos , Endopeptidases/isolamento & purificação , Proteínas Ligantes de Maltose/isolamento & purificação , Peptídeos/química , Plasmídeos/metabolismo , Dióxido de Silício/química , Marcadores de Afinidade/química , Cromatografia de Afinidade/instrumentação , Clonagem Molecular , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Peptídeos/metabolismo , Plasmídeos/química , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/química , Coloração e Rotulagem/métodosRESUMO
The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.