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1.
Chem Commun (Camb) ; 55(60): 8880-8883, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31321399

RESUMO

We developed an artificial hydrolase based on the symmetrical Pizza6 ß-propeller protein for the metal-free hydrolysis of 4-nitrophenyl acetate and butyrate. Through site-specific mutagenesis and crystallisation studies, the catalytic mechanism was investigated and found to be dependent on a threonine-histidine dyad. The mutant with additional histidine residues generated the highest kcat values, forming a His-His-Thr triad and matched previously reported metalloenzymes. The highly symmetrical ß-propeller artificial enzymes and their protein-metal complexes have potential to be utilised in bioinorganic and supramolecular chemistry, as well as being developed further into 2D/3D catalytic materials.


Assuntos
Hidrolases/química , Sequência de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Butiratos/química , Catálise , Cobre/química , Histidina/química , Histidina/genética , Hidrolases/genética , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Nitrofenóis/química , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Treonina/química , Zinco/química
2.
Chem Commun (Camb) ; 55(61): 8959-8962, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290487

RESUMO

Hydrocarbon stapled peptides are promising therapeutics for inhibition of intracellular protein-protein interactions. Here we develop a new high-throughput strategy for hydrocarbon stapled peptide discovery based on mRNA display of peptides containing α-methyl cysteine and cyclized with m-dibromoxylene. We focus on development of a peptide binder to the HPV16 E2 protein.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Evolução Molecular Direcionada/métodos , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Fatores de Transcrição/metabolismo , Alquilação , Sequência de Aminoácidos , Ciclização , Cisteína/química , Hidrocarbonetos Bromados/química , Biblioteca de Peptídeos , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/química
3.
DNA Cell Biol ; 38(8): 773-785, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31339741

RESUMO

Pierisin-5 protein (pie-5) belongs to a family of proteins possessing DNA-dependent ADP-ribosyltransferase activity, which can induce apoptotic cell death. The baculovirus-mediated expression vector system (BEVS) has been commonly used for in vitro expression of heterologous protein subunits for basic scientific research, in addition to the development and production of diagnostics and vaccines. In this study, a new method for the in vitro expression of the cytotoxic protein was established using the baculovirus expression system. The antiproliferative and apoptotic effect of the novel recombinant pierisin-5 protein (rpie-5) was investigated in different human cancer cell lines, such as HeLa, HepG2, and AGS. Cloning, in vitro overexpression, and purification of the rpie-5 protein were performed by using BEVS in Sf21 (Spodoptera frugiperda) insect cell line. The rpie-5 protein exhibits cytotoxicity in all the cell lines, but HeLa (IC50 0.6 µg/mL) was more sensitive when compared with HepG2 (IC50 1.9 µg/mL) and AGS (IC50 3.7 µg/mL) cell lines. The cytotoxic effects of rpie-5 lead to apoptotic cell death in cancer cells and resulted in nuclear fragmentation, enlargement of the nucleus, loss of mitochondrial membrane potential, and finally release of lactose dehydrogenase (LDH) enzyme from the cell membrane. This study reports the molecular mechanism of apoptotic cell death through the upregulation of Bax (Bcl-2 family activating protein-X), Bad, APAF-1 (apoptotic protease activating factor-1), Cyt-c, and caspase-3/9 and the downregulation of Bcl-2 (B-cell lymphoma 2) in rpie-5-treated cancer cells. The study concludes that rpie-5 has p53-independent apoptosis in HepG2 cells and p53-dependent apoptosis in HeLa and AGS cell lines. In the future, this study helps to understand the molecular mechanism of rpie-5 to induction of apoptosis and cell death.


Assuntos
ADP Ribose Transferases/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Proteínas Recombinantes/farmacologia , ADP Ribose Transferases/genética , Animais , Apoptose/fisiologia , Baculoviridae/genética , Linhagem Celular Tumoral , Clonagem Molecular , Humanos , Proteínas de Insetos/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/genética , Células Sf9 , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética
4.
Chem Biol Interact ; 310: 108756, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325422

RESUMO

Human butyrylcholinesterase (BChE) is a widely distributed plasma enzyme. For decades, numerous research efforts have been directed at engineering BChE as a bioscavenger of organophosphorus insecticides and chemical warfare nerve agents. However, it has been a grand challenge to cost-efficiently produce BChE in large-scale. Recently reported studies have successfully designed a truncated BChE mutant (with amino-acid substitutions on 47 residues that are far away from the catalytic site), denoted as BChE-M47 for convenience, which can be expressed in E. coli without loss of its catalytic activity. In this study, we aimed to dimerize the truncated BChE mutant protein expressed in a prokaryotic system (E. coli) in order to further improve its thermal stability by introducing a pair of cross-subunit disulfide bonds to the BChE-M47 structure. Specifically, the E377C/A516C mutations were designed and introduced to BChE-M47, and the obtained new protein entity, denoted as BChE-M48, with a pair of cross-subunit disulfide bonds indeed exists as a dimer with significantly improved thermostability and unaltered catalytic activity and reactivity compared to BChE-M47. These results provide a new strategy for optimizing protein stability for production in a cost-efficient prokaryotic system. Our enzyme, BChE-M48, has a half-life of almost one week at a 37°C, suggesting that it could be utilized as a highly stable bioscavenger of OP insecticides and chemical warfare nerve agents.


Assuntos
Butirilcolinesterase/metabolismo , Engenharia de Proteínas/métodos , Butirilcolinesterase/genética , Substâncias para a Guerra Química/metabolismo , Dimerização , Estabilidade Enzimática , Escherichia coli/genética , Humanos , Inseticidas/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Compostos Organofosforados/metabolismo
5.
Adv Exp Med Biol ; 1140: 237-250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347051

RESUMO

Covalent modification of proteins is extensively used in research and industry for biosensing, medical diagnostics, targeted drug delivery, and many other practical applications. The conventional method for production of protein conjugates has changed little in the last 20 years mostly relying on reactions of side chains of cysteine and lysine residues. Due to the presence of large numbers of similar reactive amino acid residues in proteins, common synthetic methods generally produce complex mixtures of products, which are difficult to separate. An emerging alternative technology for covalent modification of proteins involves formation of a covalent bond with a hexahistidine affinity tag present in a majority of recombinant proteins without interfering with other amino acid residues. The approach is based on formation of a ternary complex of the hexahistidine sequence with a bivalent metal cation chelated by ligand bearing an electrophilic Baylis-Hillman ester group capable of subsequent formation of a covalent bond with one of the histidine residues of the tag. The reaction proceeds under mild reaction conditions in neutral aqueous solutions under high dilutions (10-5 to 10-4 M) providing a stable covalent bond between the label and an imidazole residue in a hexahistidine tag at either C- or N-terminus. Because hexahistidine affinity tag methodology is a de-facto standard for preparation of recombinant proteins our approach can be easily implemented for selective derivatization of these proteins with fluorescent groups, alkynyl groups for "click" reactions, or biotinylation.


Assuntos
Histidina/química , Oligopeptídeos/química , Engenharia de Proteínas/métodos , Indicadores e Reagentes , Proteínas Recombinantes
6.
Nat Commun ; 10(1): 2775, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235796

RESUMO

The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization.


Assuntos
Biocatálise , Endopeptidases/metabolismo , Proteínas de Plantas/metabolismo , Poliproteínas/síntese química , Engenharia de Proteínas/métodos , Estudos de Viabilidade , Microscopia de Força Atômica/métodos , Oldenlandia , Poliproteínas/genética , Poliproteínas/isolamento & purificação , Poliproteínas/ultraestrutura , Potyvirus , Desdobramento de Proteína , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Rubredoxinas/síntese química , Rubredoxinas/genética , Rubredoxinas/isolamento & purificação , Rubredoxinas/ultraestrutura , Imagem Individual de Molécula/métodos , Análise Espectral/métodos , Proteínas Virais
7.
Chem Commun (Camb) ; 55(54): 7752-7755, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31204733

RESUMO

Metal-binding peptides are versatile building blocks in supramolecular chemistry. We recently reported a class of crystalline materials formed through a combination of coiled-coil peptide self-association and metal coordination. Here, we probe the serendipitously discovered metal binding motif that drives the assembly and apply these insights to exert rational control over structure and morphology in the materials.


Assuntos
Metaloproteínas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Cobre/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Metaloproteínas/síntese química , Engenharia de Proteínas/métodos , Multimerização Proteica , Piridinas/química
9.
Biochemistry (Mosc) ; 84(Suppl 1): S32-S50, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31213194

RESUMO

High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (~650-900 nm) open a possibility for in vivo imaging of biological processes at the micro- and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes - natural NIR light-absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded fluorescent probes, similarly to GFP-like fluorescent proteins. In this review, we discuss photophysical and biochemical properties of NIR fluorescent proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR fluorescent probes are discussed. Applications of NIR fluorescent proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole-body imaging in vivo, are described. We specifically focus on the use of NIR fluorescent probes in advanced imaging technologies that combine fluorescence and bioluminescence methods with photoacoustic tomography.


Assuntos
Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Proteínas Luminescentes/química , Fitocromo/química , Engenharia de Proteínas/métodos , Fluorescência
10.
Nat Chem ; 11(7): 653-661, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182822

RESUMO

Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l-1. This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications.


Assuntos
Peptídeo Sintases/biossíntese , Engenharia de Proteínas/métodos , Aminoácidos/química , Bacillus/genética , Sequência de Bases , Escherichia coli/genética , Família Multigênica , Biblioteca de Peptídeos , Peptídeo Sintases/química , Peptídeo Sintases/genética , Photorhabdus/enzimologia , Domínios Proteicos/genética , Especificidade por Substrato , Xenorhabdus/genética
11.
Nat Chem ; 11(7): 605-614, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209296

RESUMO

Fractal topologies, which are statistically self-similar over multiple length scales, are pervasive in nature. The recurrence of patterns in fractal-shaped branched objects, such as trees, lungs and sponges, results in a high surface area to volume ratio, which provides key functional advantages including molecular trapping and exchange. Mimicking these topologies in designed protein-based assemblies could provide access to functional biomaterials. Here we describe a computational design approach for the reversible self-assembly of proteins into tunable supramolecular fractal-like topologies in response to phosphorylation. Guided by atomic-resolution models, we develop fusions of Src homology 2 (SH2) domain or a phosphorylatable SH2-binding peptide, respectively, to two symmetric, homo-oligomeric proteins. Mixing the two designed components resulted in a variety of dendritic, hyperbranched and sponge-like topologies that are phosphorylation-dependent and self-similar over three decades (~10 nm-10 µm) of length scale, in agreement with models from multiscale computational simulations. Designed assemblies perform efficient phosphorylation-dependent capture and release of cargo proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Fractais , Agregados Proteicos , Proteínas Recombinantes de Fusão/metabolismo , Algoritmos , Proteínas de Bactérias/genética , Escherichia coli/química , Humanos , Modelos Químicos , Modelos Moleculares , Fosforilação , Engenharia de Proteínas/métodos , Multimerização Proteica , Proteínas Recombinantes de Fusão/genética , Domínios de Homologia de src/genética , Quinases da Família src/metabolismo
12.
Nat Commun ; 10(1): 2013, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043592

RESUMO

Tight control over protein degradation is a fundamental requirement for cells to respond rapidly to various stimuli and adapt to a fluctuating environment. Here we develop a versatile, easy-to-handle library of destabilizing tags (degrons) for the precise regulation of protein expression profiles in mammalian cells by modulating target protein half-lives in a predictable manner. Using the well-established tetracycline gene-regulation system as a model, we show that the dynamics of protein expression can be tuned by fusing appropriate degron tags to gene regulators. Next, we apply this degron library to tune a synthetic pulse-generating circuit in mammalian cells. With this toolbox we establish a set of pulse generators with tailored pulse lengths and magnitudes of protein expression. This methodology will prove useful in the functional roles of essential proteins, fine-tuning of gene-expression systems, and enabling a higher complexity in the design of synthetic biological systems in mammalian cells.


Assuntos
Sequência de Aminoácidos/genética , Regulação da Expressão Gênica , Engenharia de Proteínas/métodos , Proteólise , Biotecnologia/métodos , Células HEK293 , Meia-Vida , Células HeLa , Humanos , Microscopia Intravital/métodos , Células-Tronco Mesenquimais , Microscopia de Fluorescência , Biologia Sintética/métodos
13.
Nat Plants ; 5(5): 505-511, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036912

RESUMO

The engineering of plant genomes presents exciting opportunities to modify agronomic traits and to produce high-value products in plants. Expression of foreign proteins from transgenes in the chloroplast genome offers advantages that include the capacity for prodigious protein output, the lack of transgene silencing and the ability to express multicomponent pathways from polycistronic mRNA. However, there remains a need for robust methods to regulate plastid transgene expression. We designed orthogonal activators that boost the expression of chloroplast transgenes harbouring cognate cis-elements. Our system exploits the programmable RNA sequence specificity of pentatricopeptide repeat proteins and their native functions as activators of chloroplast gene expression. When expressed from nuclear transgenes, the engineered proteins stimulate the expression of plastid transgenes by up to ~40-fold, with maximal protein abundance approaching that of Rubisco. This strategy provides a means to regulate and optimize the expression of foreign genes in chloroplasts and to avoid deleterious effects of their products on plant growth.


Assuntos
Proteínas de Arabidopsis/genética , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas/genética , Genes de Troca/genética , Engenharia de Proteínas , Transgenes/genética , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/genética
14.
Nat Plants ; 5(5): 486-490, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036913

RESUMO

Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers1-3. Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulates up to 1.3% of the total soluble protein, a 60-fold increase compared with previous studies2 (0.02%). This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.


Assuntos
Proteínas de Plantas/genética , Plastídeos/genética , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/genética , Transgenes/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Fluorescência Verde/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/genética , Zea mays/genética , Zea mays/metabolismo
15.
Nat Protoc ; 14(6): 1863-1883, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31076662

RESUMO

Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light-oxygen-voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/efeitos da radiação , Engenharia de Proteínas/métodos , Sítio Alostérico/efeitos dos fármacos , Sítio Alostérico/efeitos da radiação , Animais , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/efeitos da radiação , Linhagem Celular , Clonagem Molecular/métodos , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligantes , Luz , Camundongos , Modelos Moleculares , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Sirolimo/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
16.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052154

RESUMO

The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.


Assuntos
Toxinas Bacterianas/química , Engenharia de Proteínas/métodos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Nanopartículas/química , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/química , Streptococcus pyogenes/metabolismo
17.
Prep Biochem Biotechnol ; 49(8): 735-743, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31135267

RESUMO

Type I interferons (IFNs) are homologous cytokines that bind to a cell surface receptor and establish signaling pathways that motivate immune responses. The purpose of the current study is to assess the activity of a novel-engineered IFN-α2b. The crystallographic structure of IFN-α2b and its receptors was acquired from Protein Data Bank. Various amino acid substitutions were designed based on structural properties and other biological characteristics of residues to find the most effective amino acid on IFN affinity to advanced activities. The IFN-α2b mutants and receptors have been modeled and the interactions between two proteins have been studied as in silico by protein-protein docking for both mutants and native forms. The proper nucleic acid sequence IFN-α2 (T79Q) has been prepared based on the selected mutant. The modified IFN gene was cloned in pcDNA 3.1(-) and introduced to Chinese Hamster Ovary (CHO) cell line. Antiviral and antiproliferative assays of native and IFN-α2 (T79Q) proteins were performed in vitro. The results showed two-fold increasing in IFN-α2 (T79Q) activity (antiviral and antiproliferative activity) in comparison to native IFN-α2b. This engineered IFN-α2b may have significant novel therapeutic applications and in silico studies can be an influential method for practical research function and structure of these molecules.


Assuntos
Substituição de Aminoácidos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Engenharia de Proteínas , Receptores de Interferon/metabolismo , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Células CHO , Proliferação de Células/efeitos dos fármacos , Cricetulus , Células HeLa , Humanos , Interferon-alfa/química , Interferon-alfa/farmacologia , Simulação de Acoplamento Molecular , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia
18.
Appl Microbiol Biotechnol ; 103(12): 4733-4739, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31049622

RESUMO

Despite enormous progress in protein engineering, complemented by bioprocess engineering, the revolution awaiting the application of biocatalysis in the fine chemical industry has still not been fully realized. In order to achieve that, further research is required on several topics, including (1) rapid methods for protein engineering using machine learning, (2) mathematical modelling of multi-enzyme cascade processes, (3) process standardization, (4) continuous process technology, (5) methods to identify improvements required to achieve industrial implementation, (6) downstream processing, (7) enzyme stability modelling and prediction, as well as (8) new reactor technology. In this brief mini-review, the status of each of these topics will be briefly discussed.


Assuntos
Biocatálise , Biotecnologia/métodos , Engenharia de Proteínas/métodos , Estabilidade Enzimática , Aprendizado de Máquina , Modelos Teóricos
19.
Analyst ; 144(12): 3765-3772, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31089611

RESUMO

Investigation of the functions of insulin-secreting cells in response to glucose in single-living cells is essential for improving our knowledge on the pathogenesis of diabetes. Therefore, it is desired to develop a new convenient method that enables the direct detection of insulin secreted from single-living cells. Here, insulin-sensor-cells expressing a protein-based insulin-detecting probe immobilized on the extracellular membrane were developed to evaluate the insulin-secretion response in single-living pancreatic ß cells. The protein-based insulin-detecting probe (NαLY) was composed of a bioluminescent protein (nano-luc), the αCT segment of the insulin receptor, L1 and CR domains of the insulin receptor, and a fluorescent protein (YPet). NαLY exhibited a bioluminescence resonance energy transfer (BRET) signal in response to insulin; thus, cells of Hepa1-6 line were genetically engineered to express NαLY on the extracellular membrane. The cells were found to act as insulin-sensor-cells, exhibiting a BRET signal in response to insulin. When the insulin-sensor-cells and pancreatic ß cells (MIN6 cell line) were cocultured and stimulated with glucose, insulin-sensor-cells nearby pancreatic ß cells showed the spike-shaped BRET signal response, whereas the insulin-sensor-cells close to one pancreatic ß cell did not exhibit such signal response. However, all the insulin-sensor-cells showed a gradual increase in BRET signals, which were presumably attributed to the increase in insulin concentrations in the culture dish, confirming the function of these insulin-sensor-cells. Therefore, we demonstrated that heterogenetic insulin secretion in single-living pancreatic ß cells could be measured directly using the insulin sensor cells.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Células Secretoras de Insulina/metabolismo , Insulina/análise , Análise de Célula Única/métodos , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Fluorescência , Glucose/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Engenharia de Proteínas/métodos , Receptor de Insulina/genética , Receptor de Insulina/metabolismo
20.
Prep Biochem Biotechnol ; 49(5): 529-534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31030612

RESUMO

Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD+ dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD+ to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH.


Assuntos
Chaetomium/enzimologia , Formiato Desidrogenases/química , Proteínas Fúngicas/química , Histidina/química , Proteínas Recombinantes/química , Catálise , Ensaios Enzimáticos , Formiato Desidrogenases/genética , Formiato Desidrogenases/isolamento & purificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Histidina/genética , Domínios Proteicos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Solubilidade
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