RESUMEN
Two angiostatic fusion proteins (hAE and hEA) of human angiostatin (hAS) and endostatin (hES) proteins differed in tandem connection manner were constructed and evaluated for synergistic anti-angiogenic effects. The 65 kDa secreted fusion proteins from Pichia pastoris expression were verified by mass-spec analysis and western blotting assay. Luciferase reporter gene assay using VEGF promoter revealed that angiostatin-endostatin fusion protein (hAE) and its corresponding fusion gene delivery on Human Microvascular Endothelial Cells (HMEC-1) resulted in more potent synergistic anti-angiogenic effects than endostatin-angiostatin fusion protein (hEA). These facts suggest that the orientation of fusion genes between hAS and hES might be an important factor for developing therapeutic proteins.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Angiostatinas/genética , Angiostatinas/farmacología , Endostatinas/genética , Endostatinas/farmacología , Expresión Génica , Inhibidores de la Angiogénesis/genética , Inhibidores de la Angiogénesis/metabolismo , Angiostatinas/metabolismo , Sinergismo Farmacológico , Endostatinas/metabolismo , Células Endoteliales/efectos de los fármacos , Humanos , Pichia/genética , Pichia/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Among human antimicrobial peptides (hAMPs), DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. It offers a promising alternative to conventional antibiotics. The 458-bp-long dermcidin cDNA was amplified by PCR using a human fetal cDNA library as a template. The 147-bp fragment of the MDCD-1L gene encoding an additional methionine residue was subcloned into the pTYB11 vector. Recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads. A small peptide with a molecular mass of about 5 kDa was detected by tricine gel electrophoresis. The recombinant MDCD-1L peptide was purified from the gel and its amino acid sequence was determined by nanoLC-ESI-MS/MS analysis. The initiating amino acid, methionine, remained attached to the N-terminal region of recombinant MDCD-1L. Purified MDCD-1L showed antimicrobial activity against a Micrococcus luteus test strain.
Asunto(s)
Antibacterianos/aislamiento & purificación , Escherichia coli/genética , Expresión Génica , Inteínas , Péptidos/genética , Péptidos/aislamiento & purificación , Antibacterianos/metabolismo , Antibacterianos/farmacología , Escherichia coli/metabolismo , Humanos , Micrococcus/efectos de los fármacos , Péptidos/metabolismo , Péptidos/farmacología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
For making efficient bioelectronic device, we have developed novel immobilization method of cupredoxin azurin modified on gold (Au) surface. A recombinant protein with cysteine residue by using site-directed mutagenesis was designed and then directly immobilized on Au surface without any chemical linker. The immobilization of the functionalized protein is confirmed by surface plasmon resonance (SPR) and its surface morphology is analyzed by scanning tunneling microscopy (STM). The immobilization efficiency has been increased about 75.6%, as compared to that of wild-type azurin. The electrochemical property of the fabricated thin film was investigated by the cyclic voltammetry (CV). As a result, cysteine-modified azurin can be used for making high-quality protein film, and applied to the fabrication of nano-scale bioelectronics.
Asunto(s)
Azurina , Oro/química , Microscopía de Túnel de Rastreo/métodos , Mutagénesis Sitio-Dirigida/métodos , Resonancia por Plasmón de Superficie/métodos , Adsorción , Azurina/química , Azurina/genética , Electroquímica , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genéticaRESUMEN
The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.
Asunto(s)
Péptidos Catiónicos Antimicrobianos , Micrococcus luteus/citología , Micrococcus luteus/efectos de los fármacos , Pichia/metabolismo , Ingeniería de Proteínas/métodos , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Humanos , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , CatelicidinasRESUMEN
In order to evaluate the role of transforming growth factor (TGF)-beta3 in the neurodegenerative process, we examined the levels of mRNA and immunocytochemical distribution for TGF-beta3 in the rat hippocampus after systemic kainic acid (KA) administration. Hippocampal TGF-beta3 mRNA level was reduced 3 h after KA injection. However, the levels of TGF-beta3 mRNA were elevated 1 day post-KA and lasted for at least 30 days. A mild TGF-beta3 immunoreactivity (TGF-beta3-IR) in the Ammon's horn and a moderate TGF-beta3-IR in the dentate granule cells were observed in the normal hippocampus. The CA1 and CA3 neurons lost their TGF-beta3-IR, while TGF-beta3-positive glia-like cells proliferated mainly throughout the CA1 sector and had an intense immunoreactivity at 7, 15 and 30 days after KA. This immunocytochemical distribution of TGF-beta3-positive non-neuronal populations was similar to that of glial fibrillary acidic protein (GFAP)-positive cells. Double labeling immunocytochemical analysis demonstrated colocalization of TGF-beta3- and GFAP-immunoreactivity in the same cells. These findings suggest a compensatory mechanism of astrocytes for the synthesis of TGF-beta3 protein in response to KA-induced neurodegeneration. In addition, exogenous TGF-beta3 (5 or 10 ng/i.c.v.) significantly attenuated KA-induced seizures and neuronal damages in a dose-related manner. Therefore, our results suggest that TGF-beta3 plays an important role in protective mechanisms against KA-induced neurodegeneration.