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1.
PLoS One ; 15(8): e0237295, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756606

RESUMO

We develop fully glycosylated computational models of ACE2-Fc fusion proteins which are promising targets for a COVID-19 therapeutic. These models are tested in their interaction with a fragment of the receptor-binding domain (RBD) of the Spike Protein S of the SARS-CoV-2 virus, via atomistic molecular dynamics simulations. We see that some ACE2 glycans interact with the S fragments, and glycans are influencing the conformation of the ACE2 receptor. Additionally, we optimize algorithms for protein glycosylation modelling in order to expedite future model development. All models and algorithms are openly available.


Assuntos
Betacoronavirus/metabolismo , Simulação de Dinâmica Molecular , Peptidil Dipeptidase A/química , Glicoproteína da Espícula de Coronavírus/química , Algoritmos , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Glicosilação , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo
2.
Arch Virol ; 165(10): 2301-2309, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32757056

RESUMO

Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs) in vitro and contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6× His tag using baculovirus-infected silkworm larvae as well as the E. coli BL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae and E. coli BL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPs in vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.


Assuntos
Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Bombyx/virologia , Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Baculoviridae/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Circovirus/genética , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/imunologia , Soros Imunes/química , Imunogenicidade da Vacina , Larva/virologia , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificação
3.
PLoS One ; 15(8): e0237295, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-695314

RESUMO

We develop fully glycosylated computational models of ACE2-Fc fusion proteins which are promising targets for a COVID-19 therapeutic. These models are tested in their interaction with a fragment of the receptor-binding domain (RBD) of the Spike Protein S of the SARS-CoV-2 virus, via atomistic molecular dynamics simulations. We see that some ACE2 glycans interact with the S fragments, and glycans are influencing the conformation of the ACE2 receptor. Additionally, we optimize algorithms for protein glycosylation modelling in order to expedite future model development. All models and algorithms are openly available.


Assuntos
Betacoronavirus/metabolismo , Simulação de Dinâmica Molecular , Peptidil Dipeptidase A/química , Glicoproteína da Espícula de Coronavírus/química , Algoritmos , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Glicosilação , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
Int J Mol Sci ; 21(11)2020 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-32503354

RESUMO

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a "lectibody". Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.


Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Lectinas/metabolismo , Trans-Splicing , Brevibacillus/imunologia , Clorófitas/metabolismo , HIV/fisiologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Lectinas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Vírus da SARS/fisiologia , Internalização do Vírus/efeitos dos fármacos
5.
PLoS One ; 15(5): e0233794, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470013

RESUMO

The domestic ferret (Mustela putorius furo) provides a critical animal model to study human respiratory diseases. However immunological insights are restricted due to a lack of ferret-specific reagents and limited genetic information about ferret B and T cell receptors. Here, variable, diversity and joining genes within the ferret kappa, lambda and heavy chain immunoglobulin loci were annotated using available genomic information. A multiplex PCR approach was derived that facilitated the recovery of paired heavy and light chain immunoglobulin sequences from single sorted ferret B cells, allowing validation of predicted germline gene sequences and the identification of putative novel germlines. Eukaryotic expression vectors were developed that enabled the generation of recombinant ferret monoclonal antibodies. This work advances the ferret as an informative immunological model for viral diseases by allowing the in-depth interrogation of antibody-based immunity.


Assuntos
Linfócitos B/imunologia , Modelos Animais de Doenças , Furões , Cadeias Leves de Imunoglobulina/genética , Receptores de Antígenos de Linfócitos B/genética , Animais , Anticorpos Monoclonais/biossíntese , Linfócitos B/citologia , Sequência de Bases , Furões/genética , Furões/imunologia , Genoma , Proteínas Recombinantes de Fusão/biossíntese
6.
Mol Biotechnol ; 62(3): 200-209, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32030628

RESUMO

Tryptophan hydroxylase-type 2 (Tph2) is the first rate-limiting step in the biosynthesis of serotonin (5-HT) in the brain. The ophthalmic administration (Op-Ad) is a non-invasive method that allows delivering genetic vehicles through the eye and reaches the brain. Here, the murine Tph2 gene was cloned in a non-viral vector (pIRES-hrGFP-1a), generating pIRES-hrGFP-1a-Tph2, plus the FLAG-tag. Recombinant Tph2-FLAG was detected and tested in vitro and in vivo, where 25 µg of pIRES-hrGFP-1a-Tph2-FLAG was Op-Ad to mice. The construct was capable of expressing and producing the recombinant Tph2-FLAG in vitro and in vivo. The in vivo assays showed that the construct efficiently crossed the Hemato-Ocular Barrier and the Blood-Brain Barrier, reached brain cells, passed the optical nerves, and transcribed mRNA-Tph2-FLAG in different brain areas. The recombinant Tph2-FLAG was observed in amygdala and brainstem, mainly in raphe dorsal and medial. Relative Tph2 expression of threefold over basal level was recorded three days after Op-Ad. These results demonstrated that pIRES-hrGFP-Tph2-FLAG, administrated through the eyes was capable of reaching the brain, transcribing, and translating Tph2. In conclusion, this study showed the feasibility of delivering therapeutic genes, such as the Tph2, the first enzyme, rate-limiting step in the 5-HT biosynthesis.


Assuntos
Barreira Hematoencefálica/metabolismo , Expressão Gênica , Nervo Óptico/metabolismo , Plasmídeos , Proteínas Recombinantes de Fusão , Triptofano Hidroxilase , Administração Oftálmica , Animais , Barreira Hematoencefálica/citologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nervo Óptico/citologia , Plasmídeos/genética , Plasmídeos/farmacocinética , Plasmídeos/farmacologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Triptofano Hidroxilase/biossíntese , Triptofano Hidroxilase/genética
7.
Protein Expr Purif ; 169: 105587, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32001359

RESUMO

Prs (phosphoribosyl pyrophosphate synthase) is a broadly conserved protein that synthesises 5-phosphoribosyl 1-pyrophospate (PRPP); a substrate for biosynthesis of at least 10 enzymatic pathways including biosynthesis of DNA building blocks - purines and pyrimidines. In Escherichia coli, it is a protein of homo-hexameric quaternary structure, which can be challenging to work with, due to frequent aggregation and activity loss. Several studies showed brief purification protocols for various bacterial PRPP synthases, in most cases involving ammonium sulfate precipitation. Here, we provide a protocol for expression of E. coli Prs protein in Rosetta (DE3) and BL21 (DE3) pLysE strains and a detailed method for His-Prs and untagged Prs purification on nickel affinity chromatography columns. This protocol allows purification of proteins with high yield, purity and activity. We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting the proteins purified with our protocol resemble their physiological state. The protocol for Prs purification showed here indicates guidance to improve stability and quality of the protein and to ensure more reliable results in further assays in-vitro.


Assuntos
Fosforribosil Pirofosfato/biossíntese , Proteínas Recombinantes de Fusão , Cromatografia de Afinidade , Clonagem Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Fosforribosil Pirofosfato/química , Fosforribosil Pirofosfato/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Temperatura
8.
Protein Expr Purif ; 169: 105568, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31935447

RESUMO

About half a century after antibiotics discovery, multi-antibiotic-resistant bacteria posed a new challenge to medicine. Attempts to discover new antibiotics have drawn the attention to Antimicrobial Peptides (AMPs). The rapid growth, besides its known genetic and manipulation systems, makes E. coli the preferred host system for production of recombinant proteins on an industrial scale. To produce AMPs in E. coli, the application of fusion-tags with the aim of stability, solubility, and prevention of antimicrobial activity is one of the best practices in this regard. In this study, we presented two different expression systems for the production of PR-39 in E. coli; one in fusion with intein-Chitin binding domain (CBD) and another in fusion with SUMO accompanied by polyhistidine affinity tag. Both were cloned in the NdeI-XhoI sites of pET-17b and transformed to E. coli BL21 (DE3) pLysS. Recombinant bacteria were cultured and induced with 0.4 mM IPTG at 30 °C. Expression and purification of target proteins were confirmed by Tricine- SDS-PAGE and dot blot analysis. Recovery of 250 µg PR-39/L from SUMO fusion system and 280 µg PR-39/L from the intein fusion system was achieved. Both purified peptides showed antibacterial activity using MIC/MBC demonstrating their functionality after SUMO and intein mediated purification.


Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Escherichia coli , Proteínas Recombinantes de Fusão/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Inteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética
9.
J Biol Chem ; 295(10): 3189-3201, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31980459

RESUMO

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of ß- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein-Barr virus (EBV) BFRF1-BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approximately 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these ß- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.


Assuntos
Betaherpesvirinae/metabolismo , Gammaherpesvirinae/metabolismo , Proteínas Virais/química , Sequência de Aminoácidos , Cristalografia por Raios X , Citomegalovirus/metabolismo , Células HeLa , Herpesvirus Humano 4/metabolismo , Humanos , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Ressonância de Plasmônio de Superfície , Proteínas Virais/genética , Proteínas Virais/metabolismo
10.
Mater Sci Eng C Mater Biol Appl ; 106: 110145, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753333

RESUMO

There has been a significant increase in the use of sensitive biological components, e.g., growth factors or enzymes, in implanted scaffolds/devices. To prevent diffusion away from the targeted area and to maximize access of the biological agent to the desired target, it is necessary to provide a supportive substrate to immobilize and protect biological agents from the environment. For this purpose, nanofiber fabrics are highly promising due to their high porosity, capacity for solution flow-through and high surface-to-volume ratio. However, electrospinning often requires harsh processing conditions, such as the use of volatile solutions, which can result in loss of activity of the incorporated biological components. In this study we developed a mild process for electrospinning of eADF4(C16), a recombinant spider silk protein. eADF4(C16) is non-cytotoxic, displays excellent stability against hydrolytic and enzymatic degradation and opens the opportunity for genetic addition of bioactive factors. Therefore, an aqueous spinning dope of eADF4(C16) was loaded with either green fluorescence protein (GFP) or the recombinant fusion protein GFP-eADF4(C16). The fluorescence activity of GFP is dependent on its proper folding, which does not occur in organic solvents, making it an attractive model protein. We were able to demonstrate the usability as well as the significance of the all-aqueous processing conditions for the activity of GFP in electrospun spider silk scaffolds.


Assuntos
Fibroínas/química , Água/química , Animais , Fibroínas/genética , Fibroínas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Seda/metabolismo , Espectrometria de Fluorescência , Aranhas
11.
PLoS One ; 14(11): e0225624, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31756235

RESUMO

IL-21 is the most recently discovered common gamma-chain cytokine that promotes persistent T-cell responses in chronic infections, autoimmunity and cancer. However, the therapeutic potential of inhibiting the IL-21-BATF signaling axis, particularly in transplant rejection, remains unclear. We used heart transplant models to examine the effects of IL-21 blockade in prevention of chronic cardiac allograft vasculopathy (CAV) using genetic knock-out and therapeutic approaches. Both wild-type C57BL/6 and IL-21-/- strains acutely rejected Balb/c skin grafts and once immunized with this skin graft, rejected Balb/c heart allografts in an accelerated fashion. However, when transplanted with heart grafts from the class-II major histocompatibility complex mutant, B6bm12 mice; wild-type recipients developed CAV, while IL-21-/- recipients were protected, even at day 100 post-transplant. Similarly, BATF-/- recipients, lacking the transcription factor BATF responsible for IL-21 production, did not develop CAV in B6-bm12 heart allografts. Strikingly, in a transient treatment protocol, the development of CAV in wild-type recipients of B6-bm12 hearts allografts was blocked by the administration of IL-21 receptor fusion protein (R-Fc). Thus, we demonstrate that CAV is regulated at least in part by IL-21 signaling and its blockade by genetic approaches or therapy with IL-21R-Fc prevents CAV in mice.


Assuntos
Rejeição de Enxerto/etiologia , Transplante de Coração/efeitos adversos , Interleucinas/genética , Transplante Homólogo/efeitos adversos , Doenças Vasculares/etiologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Fatores de Transcrição de Zíper de Leucina Básica/genética , Rejeição de Enxerto/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Interferon gama/metabolismo , Interleucinas/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-21/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/biossíntese , Transplante de Pele/efeitos adversos , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Doenças Vasculares/prevenção & controle
12.
Biologicals ; 62: 22-26, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31668855

RESUMO

Salmonella is found to be a major causes of food borne diseases globally. Poultry products contaminated with this pathogen is one of the major sources of infections in humans. Outer membrane protein C (OmpC) of Salmonella Typhimurium is a promising DNA vaccine candidate to mitigate Salmonella infection in poultry. However, the large-scale production of bioactive recombinant OmpC (rOmpC) protein is hindered due to the formation of inclusion bodies in Escherichia coli. The objective of this work was to attain high level expression of rOmpC protein, purify and evaluate its functional properties. The ompC gene was optimized and fused with small ubiquitin-related modifier (SUMO) gene for high level expression as soluble protein. The fusion protein with ~58 kDa molecular weight was observed on SDS-PAGE gel. The expression levels of rOmpC fusion protein reached maximum of 38% of total soluble protein (TSP) after 8 h of 0.2% rhamnose induction. Protein purification was carried out using nickel nitrilotriacetic acid (Ni-NTA) purification column. Western blot were performed to analyse expression and immunoreactivity of rOmpC fusion protein. The results indicate that SUMO fusion system is ideal for large scale production of functional rOmpC fusion protein expression in E. coli.


Assuntos
Proteínas de Bactérias , Escherichia coli , Imunoglobulinas/imunologia , Porinas , Proteínas Recombinantes de Fusão , Proteína SUMO-1 , Salmonella typhimurium/genética , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Galinhas , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Porinas/biossíntese , Porinas/genética , Porinas/imunologia , Porinas/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteína SUMO-1/biossíntese , Proteína SUMO-1/genética , Proteína SUMO-1/imunologia , Proteína SUMO-1/isolamento & purificação , Salmonella typhimurium/metabolismo
13.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(4): 506-511, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31642227

RESUMO

OBJECTIVE: To analyse the immunogenicity of a fusion protein containing cell epitopes of Mycobacterium tuberculosis genes Rv2660c, Rv2460c, Rv3875 and Rv3804, and to evaluate the feasibility of using it as a novel target antigen for developing multi-stage TB vaccines. METHODS: Cell epitopes of Rv2660c, Rv2460c, Rv3875 and Rv3804c were fused in series to form a new antigen gene (named msv). Then msv was cloned into the prokaryotic expression vector pEASY-Blunt E1. The fusion protein msv was expressed by pEASY-Blunt E1 under the induction of isopropyl-ß-d-thiogalactoside (IPTG). Purified the protein by affinity chromatography and identified the protein by SDS-PAGE and Western blot. To evaluate the immunogenicity of the protein, the mice were immunized with the purified fusion protein, and the titer of the antibody in mice serum was evaluated by ELISA. Besides, splenocytes of immunized mice were separated and splenocytes proliferation was determined under the stimulation of the protein. RESULTS: The prokaryotic expression plasmid carrying msv gene was constructed successfully and msv protein could be expressed by the plasmid under the induction of IPTG. SDS-PAGE and Western blot results confirmed that a purified protein (relative molecular mass was 41.3×103) was obtained. ELISA result indicated that the titer of the antibody in msv immunized mice serum was about 1:81 920.The spleen lymphocyte proliferation assay showed that after immunization with msv protein, significant proliferation of antigen-sensitized lymphocytes was observed. CONCLUSIONS: The fusion protein msv was successfully expressed and purified, which can induce humoral and cellular immunity in mice. It may be used as an antigen component for the development of TB vaccine in the future.


Assuntos
Proteínas de Bactérias/imunologia , Epitopos/biossíntese , Mycobacterium tuberculosis , Proteínas Recombinantes de Fusão/biossíntese , Animais , Antígenos de Bactérias/imunologia , Western Blotting , Proliferação de Células , Imunidade Celular , Imunidade Humoral , Linfócitos/citologia , Camundongos , Plasmídeos , Proteínas Recombinantes de Fusão/imunologia , Baço/citologia
14.
ACS Appl Mater Interfaces ; 11(44): 41091-41099, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31600051

RESUMO

Polydimethylsiloxane (PDMS) is a synthetic material with excellent properties for biomedical applications because of its easy fabrication method, high flexibility, permeability to oxygen, transparency, and potential to produce high-resolution structures in the case of lithography. However, PDMS needs to be modified to support homogeneous cell attachments and spreading. Even though many physical and chemical methods, like plasma treatment or extracellular matrix coatings, have been developed over the last decades to increase cell-surface interactions, these methods are still very time-consuming, often not efficient enough, complex, and can require several treatment steps. To overcome these issues, we present a novel, robust, and fast one-step PDMS coating method using engineered anchor peptides fused to the cell-adhesive peptide sequence (glycine-arginine-glycine-aspartate-serine, GRGDS). The anchor peptide attaches to the PDMS surface predominantly by hydrophobic interactions by simply dipping PDMS in a solution containing the anchor peptide, presenting the GRGDS sequence on the surface available for cell adhesion. The binding performance and kinetics of the anchor peptide to PDMS are characterized, and the coatings are optimized for efficient cell attachment of fibroblasts and endothelial cells. Additionally, the applicability is proven using PDMS-based directional nanotopographic gradients, showing a lower threshold of 5 µm wrinkles for fibroblast alignment.


Assuntos
Adesão Celular , Dimetilpolisiloxanos/química , Oligopeptídeos/química , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Adesão Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Microscopia de Fluorescência , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Propriedades de Superfície
15.
Bratisl Lek Listy ; 120(10): 757-763, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31663351

RESUMO

AIM: The study was aimed at design a good fusion construct that would successfully express the recombinant proteins and produce peptides in Escherichia coli. Two different constructs including human epidermal growth factor (hEGF) gene were designed to obtain an efficient expression level of hEGF. The hEGF sequence was inserted in pET32a vector containing thioredoxin (Trx) sequence and modified pET15b vector containing intein and elastin-like polypeptide (ELP). METHODS: The vectors were transformed into E. coli TOP10F' for multiplication and further into E. coli BL21 (DE3) to express protein. The hEGF expression was induced by isopropyl ß-D-1-thiogalactopyranoside (IPTG) while the expression levels were evaluated by SDS-PAGE and western blotting and compared by ImageJ analysis, BCA and Elisa assays. RESULTS: The expression level after 2 hours of IPTG induction was significantly higher than after other induction times. ImageJ, BCA and Elisa analyses demonstrated that the Trx presence enhanced protein expression significantly when compared to ELP-intein-based construct. CONCLUSION: The pET32a-Trx-hEGF construct had a higher expression than pET15b-ELP-intein-hEGF. Overall, considering Trx, the fusion protein in construct design can make it suitable to significantly express hEGF compared to ELP-intein while its combination with ELP-intein may improve the expression of the ELP-intein construct (Tab. 2, Fig. 7, Ref. 34).


Assuntos
Elastina , Fator de Crescimento Epidérmico/biossíntese , Escherichia coli , Inteínas , Humanos , Peptídeos , Proteínas Recombinantes de Fusão/biossíntese
16.
PLoS One ; 14(9): e0221394, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31483818

RESUMO

BACKGROUND: Malaria caused by Plasmodium falciparum is one of the major threats to human health globally. Despite huge efforts in malaria control and eradication, highly effective vaccines are urgently needed, including vaccines that can block malaria transmission. Chimeric virus-like particles (VLP) have emerged as a promising strategy to develop new malaria vaccine candidates. METHODS: We developed yeast cell lines and processes for the expression of malaria transmission-blocking vaccine candidates Pfs25 and Pfs230 as VLP and VLP were analyzed for purity, size, protein incorporation rate and expression of malaria antigens. RESULTS: In this study, a novel platform for the display of Plasmodium falciparum antigens on chimeric VLP is presented. Leading transmission-blocking vaccine candidates Pfs25 and Pfs230 were genetically fused to the small surface protein (dS) of the duck hepatitis B virus (DHBV). The resulting fusion proteins were co-expressed in recombinant Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) strains along with the wild-type dS as the VLP scaffold protein. Through this strategy, chimeric VLP containing Pfs25 or the Pfs230-derived fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated from 100 g dry cell weight with a maximum protein purity of 90% on the protein level. Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported the surface display of the malaria antigens on the native VLP. CONCLUSION: The incorporation of leading Plasmodium falciparum transmission-blocking antigens into the dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. Competitive processes for efficient production and purification were established in this study.


Assuntos
Antígenos de Protozoários/metabolismo , Vírus da Hepatite B do Pato/genética , Vacinas Antimaláricas/biossíntese , Pichia/metabolismo , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Animais , Anticorpos Bloqueadores/imunologia , Antígenos de Protozoários/genética , Patos/virologia , Humanos , Malária/prevenção & controle , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/isolamento & purificação , Plasmodium falciparum/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação
17.
Cell ; 178(5): 1222-1230.e10, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31442409

RESUMO

The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer.


Assuntos
Ligantes , Receptores CCR7/metabolismo , Regulação Alostérica , Sítios de Ligação , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Neuraminidase/genética , Neuraminidase/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores CCR2/química , Receptores CCR2/metabolismo , Receptores CCR7/antagonistas & inibidores , Receptores CCR7/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação
18.
Mar Biotechnol (NY) ; 21(5): 697-706, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31372794

RESUMO

The availability of sexually mature fish often dictates the success of its captive breeding. In this study, we induced reproductive development in juvenile protogynous tiger grouper through oral administration of a plasmid (p) containing an engineered follicle-stimulating hormone (FSH). An expression construct (pcDNA3.1) was designed to express a single-chain FSH consisting of giant grouper FSH ß-subunit and glycoprotein subunit-α (CGα), linked by the carboxy-terminal peptide (CTP) sequence from the human chorionic gonadotropin (hCG). Single oral delivery of pFSH encapsulated in liposome and chitosan to tiger grouper yielded a significant increase in plasma FSH protein level after 4 days. Weekly pFSH feeding of juvenile tiger groupers for 8 weeks stimulated ovarian development as indicated by a significant increase in oocyte diameter and progression of oocytes to cortical alveolar stage. As the pFSH treatment progressed from 20 to 38 weeks, female to male sex change was initiated, characterized by oocyte regression, proliferation of spermatogonial cells, and occurrence of spermatogenic cysts. It was also associated with significantly lower mRNA expression of steroidogenic genes (cyp11b, cyp19a1a, and foxl2) and basal plasma levels of sex steroid hormones 17ß-estradiol (E2), testosterone (T), and 11-ketotestosterone (11KT). Results suggest that pFSH stimulates ovarian development up to cortical alveolar stage and then initiates sex change in tiger grouper. These findings significantly contribute to our knowledge on the role of FSH in the development of protogynous hermaphroditic fish. This study is the first to demonstrate induction of reproductive development in fish through oral delivery of plasmid gonadotropin.


Assuntos
Gonadotropina Coriônica/genética , Hormônio Foliculoestimulante/genética , Gônadas/efeitos dos fármacos , Organismos Hermafroditas/efeitos dos fármacos , Perciformes/genética , Processos de Determinação Sexual/efeitos dos fármacos , Diferenciação Sexual/efeitos dos fármacos , Administração Oral , Animais , Quitosana/química , Gonadotropina Coriônica/administração & dosagem , Gonadotropina Coriônica/biossíntese , Composição de Medicamentos , Feminino , Proteínas de Peixes/biossíntese , Proteínas de Peixes/genética , Hormônio Foliculoestimulante/administração & dosagem , Hormônio Foliculoestimulante/biossíntese , Hormônios Esteroides Gonadais/biossíntese , Hormônios Esteroides Gonadais/genética , Gônadas/crescimento & desenvolvimento , Gônadas/metabolismo , Organismos Hermafroditas/genética , Humanos , Lipossomos/administração & dosagem , Lipossomos/química , Masculino , Oogênese/efeitos dos fármacos , Oogênese/genética , Perciformes/crescimento & desenvolvimento , Perciformes/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Pré-Seleção do Sexo/métodos , Espermatogênese/efeitos dos fármacos , Espermatogênese/genética
19.
Virology ; 536: 49-57, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400549

RESUMO

Molecular adjuvants are vaccine delivery vehicle to increase specific antigens effectiveness. Herein, we concentrated on IgG Fc, an effective molecular adjuvant, to develop novel pseudorabies virus (PRV) subunit vaccines. Two major protective antigen genes of PRV were constructed and linked into the mouse IgG Fc fragment. The gD, gD-IgG2aFc, gB and gB-IgG2aFc proteins were expressed using a baculovirus system. Mice intranasally immunized with gD-IgG2aFc or gB-IgG2aFc subunit vaccine exhibited significantly higher PRV-specific antibodies, neutralizing antibodies and intracellular cytokines than the mice intranasally immunized with gD or gB subunit vaccine. Moreover, no histopathological lesions were observed in mice immunized with gB-IgG2aFc subunit vaccine via histopathology examination. Further, the gB-IgG2aFc subunit vaccine was efficient for PRV infection compared with live attenuated vaccine. Overall, these results suggest that IgG2a Fc fragment, as a potential molecular adjuvant, fused with PRV antigen might be a promising and efficient PRV vaccine candidate.


Assuntos
Herpesvirus Suídeo 1/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/biossíntese , Vacinas contra Pseudorraiva/biossíntese , Pseudorraiva/prevenção & controle , Proteínas Recombinantes de Fusão/biossíntese , Proteínas do Envelope Viral/biossíntese , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/genética , Animais , Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Baculoviridae/metabolismo , Citocinas/genética , Citocinas/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Glicoproteínas/administração & dosagem , Glicoproteínas/biossíntese , Glicoproteínas/genética , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/patogenicidade , Imunização , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/administração & dosagem , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Rim/patologia , Rim/virologia , Camundongos , Camundongos Endogâmicos BALB C , Pseudorraiva/imunologia , Pseudorraiva/mortalidade , Pseudorraiva/virologia , Vacinas contra Pseudorraiva/administração & dosagem , Vacinas contra Pseudorraiva/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Análise de Sobrevida , Suínos , Vacinas de Subunidades , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética
20.
Int J Biol Macromol ; 139: 697-711, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31381908

RESUMO

The aggregation of recombinant proteins in the different stages of purification leads to the loss of a considerable portion of target protein and reduction in the process efficiency. As the active HBsAg used in Hepatitis B vaccine production is in the form of virus-like particle (VLP), therefore the time and stages at which the VLP assembling happened through the process would be important. The aim of this study was to explore the product aggregation during different stages of large scale production of rHBsAg in Pichia pastoris at production unit of the Pasteur Institute of Iran. Dynamic light scattering (DLS) and transmission electron microscopy (TEM), and also size exclusion-high-performance liquid chromatography (SE-HPLC) were carried out on samples taken from each downstream processes steps to determine the rate of VLPs formation as the desired product and the aggregated form at each stage of the purification. Based on the results, it was found that VLPs formation started at the acid precipitation stage and reached up to 80% at the thermal treatment stage. The ultrafiltration, ion exchange chromatography and immunoaffinity chromatography stages were disclosed to have the highest contribution in the formation of VLP (virus like particle) 22 nm.


Assuntos
Antígenos de Superfície da Hepatite B/biossíntese , Vacinas contra Hepatite B/biossíntese , Agregados Proteicos , Produtos Biológicos/química , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Dissulfetos/química , Fermentação , Engenharia Genética , Vírus da Hepatite B/imunologia , Tamanho da Partícula , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Tiocianatos/química , Ultrafiltração
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