RESUMEN
A new portable molecular imprinting polymer (MIP)-SERS nanoprobe is fabricated by a convenient electrochemical method. Single-layered MoS2 is electrochemically reduced on a screen-printed electrode as the scaffold. Functional monomers o-phenylenediamine (oPD), template theophylline (THP), and SERS-active Au nanoparticles (AuNPs) are then one-step electropolymerized on the scaffold. The morphology of the nanoprobe is found to be a three-dimensional and porous structure. The abundant AuNPs with the size of 45~50 nm are trapped within the growing MIP instead of being confined to the surface. The thickness of MIP film is calculated to 25.1 nm. The nanoprobe displays a strong SERS effect for THP using 532 nm as excitation wavelength with a detection limit (LOD) of 0.01 nM. The SERS peak intensity at 1487 cm-1 increases linearly with the concentration of THP in the range 0.1 nM to 0.1 mM. After the template is removed, the imprint-removed nanoprobe is generated for selective binding of THP. The re-binding kinetics study implies the portable MIP-SERS nanoprobe can reach the adsorption equilibrium within 8 min. This nanoprobe exhibits low SERS interference for structural analogues theobromine (THB) and caffeine (CAF). The nanoprobe was employed to THP determination in tea drink samples, with recoveries ranging from 99.0 to 102.0% and relative standard deviations of < 5.0%. Graphical abstractSchematic representation of a portable molecular imprinting SERS nanoprobe used for selective and sensitive theophylline recognition. The nanoprobe is fabricated by one-step electropolymerized o-phenylenediamine (oPD), theophylline, and electroreduced Au nanoparticles (AuNPs) on reduced MoS2 (rMoS2) modified screen-printed electrode (SPE).
Asunto(s)
Técnicas Biosensibles , Disulfuros/química , Técnicas Electroquímicas , Impresión Molecular , Sondas Moleculares/química , Molibdeno/química , Teofilina/análisis , Nanopartículas/química , Oxidación-Reducción , Espectrometría RamanRESUMEN
The purpose of the present work was to develop a novel, long-acting and potent human serum albumin/granulocyte colony stimulating factor (HSA/G-CSF) therapeutic fusion protein. The novel fusion protein, called HMG, was constructed by genetically fusing mutated human derived G-CSF (mG-CSF) to the C-terminal of HSA and then prepared in Pichia pastoris. The molecular mass of HMG was about 85 kDa and the isoelectric point was 5.3. Circular dichroism spectroscopy suggested that mG-CSF retained nearly all of its native secondary structure, regardless of fusion. The binding capabilities of mG-CSF moiety to G-CSF receptor and HSA moiety to warfarin showed very little change after fusing. The bioactivity of HMG (11.0×10(6) IU/mg) was more than twice that of rHSA/G-CSF (4.6×10(6) IU/mg). A mutation was made at the 718th amino acid of HMG, substituting Ala for Thr, to investigate the glycosylation of HMG expressed in P. pastoris. Data indicated that HMG was modified at Thr718, speculatively with the addition of a mannose chain. In conclusion, a novel HSA/G-CSF fusion protein was successfully constructed based on a mutated G-CSF. This protein showed more potent bioactivity than rHSA/G-CSF and thus may be a suitable long-acting G-CSF.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/genética , Pichia/genética , Proteínas Recombinantes de Fusión/genética , Albúmina Sérica/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anticoagulantes/metabolismo , Factor Estimulante de Colonias de Granulocitos/química , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Albúmina Sérica Humana , Warfarina/metabolismoRESUMEN
The plasma half-life of therapeutic proteins is a critical factor in many clinical applications. Therefore, new strategies to prolong plasma half-life of long-acting peptides and protein drugs are in high demand. Here, we designed an artificial gelatin-like protein (GLK) and fused this hydrophilic GLK polymer to granulocyte-colony-stimulating factor (G-CSF) to generate a chimeric GLK/G-CSF fusion protein. The genetically engineered recombinant GLK/G-CSF (rGLK/G-CSF) fusion protein was purified from Pichia pastoris. In vitro studies demonstrated that rGLK/G-CSF possessed an enlarged hydrodynamic radius, improved thermal stability and retained full bioactivity compared to unfused G-CSF. Following a single subcutaneous administration to rats, the rGLK/G-CSF fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating white blood cells than G-CSF. Our findings indicate that fusion with this artificial, hydrophilic, GLK polymer provides many advantages in the construction of a potent hematopoietic factor with extended plasma half-life. This approach could be easily applied to other therapeutic proteins and have important clinical applications.
Asunto(s)
Gelatina/química , Factor Estimulante de Colonias de Granulocitos/genética , Polímeros/química , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos/efectos de los fármacos , Formación de Anticuerpos/inmunología , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estabilidad de Medicamentos , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Factor Estimulante de Colonias de Granulocitos/sangre , Factor Estimulante de Colonias de Granulocitos/química , Factor Estimulante de Colonias de Granulocitos/farmacología , Semivida , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pichia/genética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TransfecciónRESUMEN
A novel recombinant exendin-4 human serum albumin fusion protein (rEx-4/HSA) expressed in Pichia pastoris was prepared and characterized. Ex-4 is a 39-amino acid peptide isolated from the salivary gland of the lizard Heloderma suspectum and is thought to be a novel therapeutic agent for type 2 diabetes. But to gain a continued effect, the peptide has to be injected twice a day owing to its short plasma half-life (t(1/2) = 2.4 h). To extend the half-life of Ex-4 molecule in vivo, we designed a genetically engineered Ex-4/HSA fusion protein. Between Ex-4 and HSA, a peptide linker GGGGS was inserted and the fusion protein was expressed in methylotrophic yeast P. pastoris with native HSA secretion signal sequence. The recombinant protein was secreted correctly and was obtained with high purity (typically > 98%) by a three-step purification procedure. cAMP assay demonstrated that the fusion protein had a bioactivity similar to Ex-4 for interaction with GLP-1 receptors in vitro. Results from oral glucose tolerance test indicated that rEx-4/HSA could effectively improve glucose tolerance in diabetic db/db mice. Pharmacokinetics studies in cynomologus monkeys also showed that rEx-4/HSA had a much longer plasma half-life. Therefore, rEx-4/HSA fusion protein could potentially be used as a new recombinant biodrug for type 2 diabetes therapy.
Asunto(s)
Péptidos/genética , Péptidos/aislamiento & purificación , Albúmina Sérica/genética , Albúmina Sérica/aislamiento & purificación , Ponzoñas/genética , Ponzoñas/aislamiento & purificación , Animales , Secuencia de Bases , Glucemia/metabolismo , Línea Celular , Cricetinae , AMP Cíclico/biosíntesis , Cartilla de ADN/genética , Exenatida , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Hipoglucemiantes/aislamiento & purificación , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Lagartos/genética , Ratones , Ratones Endogámicos C57BL , Péptidos/farmacocinética , Péptidos/farmacología , Pichia/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacología , Albúmina Sérica/farmacocinética , Albúmina Sérica/farmacología , Transfección , Ponzoñas/farmacocinética , Ponzoñas/farmacologíaRESUMEN
Recombinant mutant human granulocyte colony stimulating factor (rmhG-CSF) was pegylated, purified and characterized. rhG-CSF was mutated in position 1,3,4,5,17, and cysteine was added in C-terminal. rmhG-CSF was pegylated by PEG-Mal 20000 and separated by ion-exchange chromatography, gel filtration chromatography. Analysis of SDS-PAGE showed thar the purity of the separated PEG-rmhG-CSF was greater than 95%. and in intro and in vivo bioactivity study showed that target modified PEG-rmhG-CSF kept full bioactivity which was better than traditional pegylation method, and longer half-life was proved in mice.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/química , Factor Estimulante de Colonias de Granulocitos/genética , Proteínas Mutantes/genética , Polietilenglicoles/química , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía por Intercambio Iónico , Factor Estimulante de Colonias de Granulocitos/biosíntesis , Humanos , Datos de Secuencia Molecular , Proteínas Mutantes/biosíntesis , Señales de Clasificación de Proteína , Proteínas RecombinantesRESUMEN
Double antibody sandwich-type ELISA was used to detect rhG-CSF in serum to study the pharmacokinetics of rhG-CSF, PEG-rhG-CSF and rHSA-hG-CSF in mice and to confirm that PEGlyation and albumin fusion of rhG-CSF technology can prolong half-life of G-CSF. Pharmacokinetic parameters were calculated with 3P87 software. T1/2 s of rhG-CSF, PEG-rhG-CSF and rHSA-hG-CSF are 2. 1 , 14.2 and 10. 6 h, respectively. T1/2 s of PEG- rhG-CSF and rHSA-hG-CSF are 7, 5 times than T1/2 s of rhG-CSF, respectively. Tpeak s of PEG-rhG-CSF and rHSA-hG-CSF are 15, 13 times than Tpeak of rhG-CSF, respectively. The result of ELISA indicates that PEGlyation and albumin fusion of rhG-CSF technology can prolong half-life of G-CSF.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/farmacocinética , Polietilenglicoles/química , Proteínas Recombinantes de Fusión/farmacocinética , Albúmina Sérica/química , Animales , Área Bajo la Curva , Ensayo de Inmunoadsorción Enzimática/métodos , Factor Estimulante de Colonias de Granulocitos/sangre , Factor Estimulante de Colonias de Granulocitos/química , Semivida , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/química , Proteínas RecombinantesRESUMEN
A long-lasting recombinant human serum albumin-interferon-alpha2b fusion protein (rHSA/IFNalpha2b) was prepared and its structure and biological activities were studied. rHSA/IFNalpha2b was expressed in methylotrophic yeast Pichia pastoris with HSA's natural signal peptide and purified by dye affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography and Sephadex G25. Purity of the prepared rHSA/IFNalpha2b was greater than 97% analyzed by non-reduced SDS-PAGE and RP-HPLC. Structure and biological activities of the prepared rHSA/IFNalpha2b were characterized by physical, chemical and biological methods. Its pI was 5.3 and showed a single band on IEF gel. Molecular weight determined by MALDI-TOF was 86004.3+/-29.2. Amino-terminal and carboxyl-terminal amino acid sequences were identical to predicted sequence. Its specific activity in vitro was 6.3+/-0.8x10(5) IU/mg fusion protein, retaining about 1.4% of that of unmodified rIFNalpha on a molar basis. After administered in monkeys, significant increases of 2',5'-oligoadenylate synthetase activity relative to IFN-alpha were maintained for 14 days in serum and the rHSA/IFNalpha2b showed more potent biological activity than IFN-alpha on a molar basis. Therefore, markedly improved in vivo biological activity of rHSA/IFNalpha2b could exhibit more potent antiviral activity than IFNalpha2b in future clinical trials.
Asunto(s)
Interferón-alfa/química , Pichia/metabolismo , Proteínas Recombinantes de Fusión/química , Albúmina Sérica/química , Secuencia de Aminoácidos , Antivirales/farmacología , Química Farmacéutica/métodos , Cromatografía , Cromatografía por Intercambio Iónico/métodos , Humanos , Interferón alfa-2 , Focalización Isoeléctrica , Datos de Secuencia Molecular , Peso Molecular , Señales de Clasificación de Proteína , Proteínas Recombinantes , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
OBJECTIVE: A mouse model of orthotopic bladder cancer simulating its human counterpart is of great importance in preclinical evaluation of new treatment modalities such as immunotxin therapy. The aim of the present study is to establish a novel nude mouse model with xenografted human bladder cancer. METHODS: Single cell suspension of an established human bladder transitional cell carcinoma (TCC) cell line BIU-87 was instilled into nude mouse bladders which were pretreated with mild acid washing. The tumor growth in mouse bladder was assessed weekly by magnetic resonance imaging (MRI). At intervals following implantation and MRI tumor detection, the animals were sacrificed for necropsy, histological examination and immunocytochemical studies. RESULTS: The overall tumor establishment was 92.9% (52/56 mice) at 7 - 36 days, while in the subgroup of animals sacrificed at 12 - 13 days, 40 out of 42 animals (95.2%) developed TCC, the majority of which was superficial. The tumor stages were assessed by gross and histopathology. Histological examination confirmed the presence of grade II - III TCC. Immunocytochemistry confirmed that the tumor model maintained the biological and immunological features of BIU-87 cells. The changes seen on MRI images well correlated with the extent of tumor invasion identified by histology. Carcinoma in situ could be detected histologically at 7 - 9 days post-inoculation and progressed into papillary or invasive tumors thereafter. CONCLUSION: The orthotopic BIU-87 TCC model in nude mice is highly reproducible and is ideal for preclinical studies on experimental intravesical therapies.