RESUMO
Disorder of complement response is a significant pathogenic factor causing some autoimmune and inflammation diseases. The Ornithodoros moubata Complement Inhibitor (OmCI), a small 17 kDa natural protein, was initially extracted from soft tick salivary glands. The protein was found binding to complement C5 specifically, inhibiting the activation of the complement pathway, which is a successful therapeutic basis of complement-mediated diseases. However, a short half-life due to rapid renal clearance is a common limitation of small proteins for clinical application. In this study, we extended the half-life of OmCI by modifying it with fatty acid, which was a method used to improve the pharmacokinetics of native peptides and proteins. Five OmCI mutants were initially designed, and single-site cysteine mutation was introduced to each of them. After purification, four OmCI mutants were obtained that showed similar in vitro biological activities. Three mutants of them were subsequently coupled with different fatty acids by nucleophilic substitution. In total, 15 modified derivatives were screened and tested for anticomplement activity in vitro. The results showed that coupling with fatty acid would not significantly affect their complement-inhibitory activity (CH50 and AH50). OmCIT90C-CM02 and OmCIT90C-CM05 were validated as the applicable OmCI bioconjugates for further pharmacokinetic assessments, and both showed improved plasma half-life in mice compared with unmodified OmCI (15.86, 17.96 vs 2.57 h). In summary, our data demonstrated that OmCI conjugated with fatty acid could be developed as the potential long-acting C5 complement inhibitor in the clinic.
Assuntos
Complemento C5 , Ácidos Graxos , Ornithodoros , Animais , Ácidos Graxos/química , Camundongos , Complemento C5/antagonistas & inibidores , Desenho de Fármacos , Meia-Vida , Proteínas Inativadoras do Complemento/farmacologia , Proteínas Inativadoras do Complemento/química , Inativadores do Complemento/farmacologia , Inativadores do Complemento/farmacocinética , Inativadores do Complemento/química , HumanosRESUMO
AA139, a variant of natural antimicrobial peptide (AMP) arenicin-3, displayed potent activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria. Nevertheless, there were currently few reports on the bioprocess of AA139, and the yields were less than 5 mg/L. Additionally, it was difficult and expensive to prepare AA139 through chemical synthesis due to its complex structure. These factors have impeded the further research and following clinical application of AA139. Here, we reported a bioprocess for the preparation of AA139, which was expressed in Escherichia coli (E. coli) BL21 (DE3) intracellularly in a soluble form via SUMO (small ubiquitin-related modifier) fusion technology. Then, recombinant AA139 (rAA139, refer to AA139 obtained by recombinant expression in this study) was obtained through the simplified downstream process, which was rationally designed in accordance with the physicochemical characteristics. Subsequently, the expression level of the interest protein was increased by 54% after optimization of high cell density fermentation (HCDF). Finally, we obtained a yield of 56 mg of rAA139 from 1 L culture with a purity of 98%, which represented the highest reported yield of AA139 to date. Furthermore, various characterizations were conducted to confirm the molecular mass, disulfide bonds, and antimicrobial activity of rAA139.
Assuntos
Peptídeos Antimicrobianos , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Peptídeos Antimicrobianos/genética , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Fermentação , Expressão GênicaRESUMO
Adrenocorticotropic hormone (ACTH) is an old medicine derived from porcine pituitary gland that has been marketed for more than 60 years. In this study, we present a recombinant approach to produce ACTH in Escherichia coli (E. coli). The SUMO-tagged fusion protein was cloned and expressed after induction with isopropyl-ß-d-thiogalactopyranoside (IPTG) at 25 °C for 8 h. The fusion protein was extracted and purified by anion exchange chromatography, and the SUMO tag was subsequently removed by digestion with ubiquitin-like protease 1 (ULP1). Approximately 95.3 mg of recombinant ACTH with 94.2% purity was obtained after cation exchange purification performed on a 5 mL column, from 286 mL fermentation broth based on the amount of pellets homogenized. The molecular mass of the recombinant ACTH was confirmed by mass spectrometry to equal 4567.32 Da.
Assuntos
Hormônio Adrenocorticotrópico , Clonagem Molecular , Expressão Gênica , Proteínas Recombinantes de Fusão , Suínos/genética , Hormônio Adrenocorticotrópico/biossíntese , Hormônio Adrenocorticotrópico/química , Hormônio Adrenocorticotrópico/genética , Hormônio Adrenocorticotrópico/isolamento & purificação , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25%) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39% activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93% activity even at 4 M NaCl. The catalytic efficiency (k cat/K m) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg(-1) s(-1) (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl2. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.
Assuntos
Proteínas de Bactérias/metabolismo , Flavobacteriaceae/enzimologia , Tolerância ao Sal , alfa-Amilases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Temperatura Baixa , Flavobacteriaceae/genética , Dados de Sequência Molecular , alfa-Amilases/química , alfa-Amilases/genéticaRESUMO
A new xylanase gene (xynA) from the marine microorganism Zunongwangia profunda was identified to encode 374 amino acid residues. Its product (XynA) showed the highest identity (42.78%) with a xylanase from Bacillus sp. SN5 among the characterized xylanases. XynA exhibited the highest activity at pH 6.5 and 30 °C, retaining 23 and 38% of the optimal activity at 0 and 5 °C, respectively. XynA was not only cold active, but also halophilic, and both its activity and thermostability could be significantly increased by NaCl, showing the highest activity (180% of the activity) at 3 M NaCl and retaining nearly 100% activity at 5 M NaCl, compared to the absence of NaCl. In the presence of 3 M NaCl, the k cat/K m value of XynA exhibited a 3.41-fold increase for beechwood xylan compared to no added NaCl, and the residual activity of XynA increased from 23% (no added NaCl) to 58% after 1 h incubation at 45 °C. This may be the first report concerning a cold-adapted xylanase from a non-halophilic species that displays the highest activity at a NaCl concentration range from 3 to 5 M. The features of cold activity and salt tolerance suggest the potential application of XynA in the food industry and bioethanol production from marine seaweeds.
Assuntos
Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Flavobacteriaceae/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Temperatura Baixa , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Flavobacteriaceae/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Tolerância ao Sal , Xilanos/metabolismoRESUMO
OBJECTIVE: To investigate the patient satisfaction with medications commonly used for migraine therapy in patients seen in headache clinic in China with emphasis on the evaluation of Chinese patent medicine (CPM) in relieving acute migraine attack. METHODS: Patients admitted at headache clinics in the neurological departments of four hospitals during April to October 2011 were enrolled in the investigation. The questionnaire was designed based on the validation of a diagnostic questionnaire for a population-based survey in China in 2009. RESULTS: Among 219 eligible patients, 58% had used CPM at the acute attack of migraine while the guideline-recommended treatments were seldom used. However, patients using CPMs were less satisfied than those using Western Medicines (WMs) in either single medication groups or mixed medication groups (P < 0.05). CONCLUSION: Fifty-eight percent of the eligible respondents in Guangdong and Guangxi Province had used CPM at the acute attack of migraine, but based on our data, the effect of CPM on treating migraine attack was poor with low satisfaction compared with WMs. However, many factors may bias or explain our findings. This suggests the need for accelerated research in understanding patient choice, treatment availability, and use of medications.
Assuntos
Analgésicos/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Transtornos de Enxaqueca/tratamento farmacológico , Satisfação do Paciente , Adulto , China , Coleta de Dados , Medicamentos de Ervas Chinesas/normas , Feminino , Humanos , MasculinoRESUMO
A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The K m and k cat values of GSxyn for ρNPX were 0.48 mM and 36.64 s−1, respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The K m and k cat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s−1, respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The K m and k cat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s−1, respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall.
Assuntos
Geobacillus stearothermophilus/enzimologia , Xilosidases/genética , Xilosidases/metabolismo , Sequência de Aminoácidos , Glicosídeos/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Especificidade por Substrato , Tirosina/genética , Xilanos/metabolismo , Xilosidases/químicaRESUMO
As an excellent fusion tag for expressing heterologous proteins, yeast SUMO (small ubiquitin-related modifier) has unique advantages such as improving solubility, promoting stability, and reducing degradation, but it lacks a simple and rapid purification method. Camelid single-domain antibodies (VHHs or nanobodies) show great promise as an efficient tool in analytical application. In this study, VHHs against SUMO protein were isolated for the first time using biopanning of an immune camelid nanobody library. Among these nanobodies, VS2 demonstrated a high expression level (1.12 g L - 1), and a high affinity for SUMO (2.26 nM). Meanwhile, VHHs were coupled to agarose resins by cysteine at the C-terminal to form affinity chromatography resins. The VS2 resin showed excellent specificity and a dynamic binding capacity for SUMO, SUMO-DsbA (disulfide oxidoreductase) and SUMO-SAM (S-adenosylmethionine synthetase) were 2.41 mg/mL resin, 7.57 mg/mL resin and 16.23 mg/mL resin, respectively. Furthermore, the VS2 resin enabled one-step purification of SUMO-fusions [SUMO-Fc (human IgG1-Fc fragment), SUMO-IGF1 (human insulin-like growth factor 1), SUMO-FGF21 (human fibroblast growth factor 21), SUMO-G-CSF (human Granulocyte colony-stimulating factor), SUMO-PDGF (human platelet-derived growth factor) and SUMO-PAS200 (conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala-and Ser)], and maintained binding capacity and selectivity over 25 purification cycles, each including 15 min of cleaning-in-place with 0.1 M NaOH. This study demonstrated that the VS2 resin was a useful tool at the laboratory scale for one-step purification of various SUMO fusions from complex mixtures.
Assuntos
Anticorpos de Domínio Único , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Humanos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Anticorpos de Domínio Único/metabolismo , Proteína SUMO-1 , Peptídeos , Saccharomyces cerevisiae/metabolismo , Cromatografia de Afinidade/métodos , Proteínas Recombinantes de FusãoRESUMO
A novel gene (BmelA) (1323bp) encoding an α-galactosidase of 440 amino acids was cloned from the deep-sea bacterium Bacillus megaterium and the protein was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of about 45 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 4, with the highest identity (74%) to α-galactosidase Mel4A from Bacillus halodurans among the characterized α-galactosidases. The recombinant BmelA displayed its maximum activity at 35 °C and pH 8.5-9.0 in 50 mM Tris-HCl buffer, and could hydrolyze different substrates with the Km values against p-nitrophenyl-α-D-galactopyranoside (pNP-α-Gal), raffinose and stachyose being 1.02±0.02, 2.24±0.11 and 3.42±0.17 mM, respectively. Besides, 4 mutants (I38 V, I38A, I38F and Q84A) were obtained by site-directed mutagenesis based on molecular modeling and sequence alignment. The kinetic analysis indicated that mutants I38 V and I38A exhibited a 1.7- and 1.4-fold increase over the wild type enzyme in catalytic efficiency (k(cat)/K(m)) against pNP-α-Gal, respectively, while mutant I38F showed a 3.5-fold decrease against pNP-α-Gal and mutant Q84A almost completely lost its activity. All the results suggest that I38 and Q84 sites play a vital role in enzyme activity probably due to their steric and polar effects on the predicted "tunnel" structure and NAD+ binding to the enzyme.
Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias/isolamento & purificação , alfa-Galactosidase/isolamento & purificação , Sequência de Aminoácidos , Bacillus megaterium/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Sequência Consenso , DNA Bacteriano/genética , Genes Bacterianos , Genoma Bacteriano , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NAD/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismoRESUMO
Pantoea agglomerans endo-ß-1, 4-mannanase (Man26P) hydrolyzes hemicellulose by cleaving the internal bonds in the mannan-based chains. In order to obtain improved enzymes and understand the structure-function relationship better, Man26P was engineered using a modified DNA shuffling method and a 96-well plate high-throughput screening technology. Compared to the wild-type enzyme, two mutants Gly267Ser and His134Arg/Phe141Leu with 1.14- and 3.30-fold increased catalytic efficiency (k(cat)/K(m)) toward locust bean gum (LBG) were obtained from the mutant library containing 19,700 clones. Combined with site-directed mutagenesis, more substitutions were introduced into the 134 site, among which, His134Arg and His134Lys exhibited 2.81- and 2.75-fold increased catalytic efficiency versus the wild-type enzyme. And with an increase in the pK(a) values of the side chains of the mutated residues at the 134 site, a decrease was observed in their K(m) values against LBG. The molecular modeling and the docking analysis of the substrate suggest that the enhancement of polar contacts or positive charges at the 134 site contribute to the interaction of the enzyme with the substrate. Furthermore, the nature of the residue at the 267 site could also significantly affect the enzyme activity by regulating substrate binding affinity and product release. The study provides useful information for the directed evolution of mannanases.
Assuntos
Proteínas de Bactérias/genética , Manosidases/genética , Pantoea/enzimologia , Proteínas Recombinantes/genética , Proteínas de Bactérias/metabolismo , Embaralhamento de DNA , Evolução Molecular Direcionada , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Cinética , Manosidases/metabolismo , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Pantoea/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Endo-ß-1, 4-xylanase was cloned from Geobacillus stearothermophilus 1A05583 by PCR. Enzymes with improved catalytic efficiency were obtained using error-prone PCR and a 96-well plate high-throughout screening system. Two variants 1-B8 and 2-H6 were screened from the mutant library containing 9000 colonies, which, when compared with the wild-type enzyme increased the catalytic efficiency (kcat/Km) by 25% and 89%, respectively, acting on beechwood xylan. By sequencing 1-B8 and 2-H6, an identical mutation point H179Y was detected and found to overlap in the active site cleft. Following the introduction of the remaining 19 amino acids into position 179 by site-saturation mutagenesis, the catalytic efficiency of H179F was found to be 3.46-fold that of the wild-type. When Whistidine was substituted by tryptophan, arginine, methionine or proline, the enzyme lost activity. Therefore, the position 179 site may play an important role in regulating the catalytic efficiency.
Assuntos
Evolução Molecular Direcionada , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Geobacillus stearothermophilus/genética , Substituição de Aminoácidos , Catálise , Domínio Catalítico/genética , Endo-1,4-beta-Xilanases/química , Geobacillus stearothermophilus/crescimento & desenvolvimento , Geobacillus stearothermophilus/metabolismo , Histidina/química , Histidina/metabolismo , Cinética , Mutagênese , Oligopeptídeos/metabolismoRESUMO
OBJECTIVE: To evaluate the feasibility of surface tractotomy of trigeminal nerve sensory root (STS) for the treatment of trigeminal neuralgia (TN). METHOD: Seven patients with TN were operated on using the STS. The six patients were followed up for 4.8-9.8 years. The trigeminal nerve root (TNR) obtained from 30 cadavers were performed microanatomical research using paraffin embedding and hematoxylin-eosin staining technique. RESULT: Clinically, the patients' symptoms, such as face ache, disappeared after the surface nerve fiber bundles of trigeminal nerve sensory root (TNSR) were cut off. Only one patient died of brainstem bleeding on postoperative day 18. Histological examination: The common type of sensory root fibers were arranged parallel for 3-6 mm at its exit of brainstem, and then the glial myelin transformed to Schwann cells. The axon bifurcated from outer layer to middle region, and gradually formed the tiny nerve fiber bundles in the surface layer and the giant nerve fiber bundles in the center of the root. CONCLUSION: TN can be radical cured by STS without lesioning of nerve functions. Therefore,this new approach is an effective, advanced surgical technique for TN treatment.