RESUMEN
With increasing worldwide rates of morbidity and mortality of pulmonary fibrosis, the development of effective therapeutics for this disease is of great interest. Secretoglobin (SCGB) 3A2, a novel cytokine-like molecule predominantly expressed in pulmonary airways epithelium, exhibits anti-inflammatory and growth factor activities. In the current study SCGB3A2 was found to inhibit TGFß-induced differentiation of fibroblasts to myofibroblasts, a hallmark of the fibrogenic process, using pulmonary fibroblasts isolated from adult mice. This induction was through increased phosphorylation of STAT1 and expression of SMAD7 and decreased phosphorylation of SMAD2 and SMAD3. To demonstrate the effect of SCGB3A2 on the TGFß signaling in vivo, a bleomycin-induced pulmonary fibrosis mouse model was used. Mice were administered bleomycin intratracheally followed by intravenous injection of recombinant SCGB3A2. Histological examination in conjunction with inflammatory cell counts in bronchoalveolar lavage fluids demonstrated that SCGB3A2 suppressed bleomycin-induced pulmonary fibrosis. Microarray analysis was carried out using RNAs from lungs of bleomycin-treated mice with or without SCGB3A2 and normal mice treated with SCGB3A2. The results demonstrated that SCGB3A2 affects TGFß signaling and reduces the expression of genes involved in fibrosis. This study suggests the potential utility of SCGB3A2 for targeting TGFß signaling in the treatment of pulmonary fibrosis.
Asunto(s)
Antibióticos Antineoplásicos/efectos adversos , Bleomicina/efectos adversos , Regulación hacia Abajo/efectos de los fármacos , Proteínas/metabolismo , Fibrosis Pulmonar/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Regulación hacia Abajo/genética , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Perfilación de la Expresión Génica , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas/genética , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Secretoglobinas , Transducción de Señal/genética , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Secretion of recombinant proteins is a common strategy for heterologous protein expression using the yeast Kluyveromyces lactis. However, a common problem is degradation of a target recombinant protein by secretory pathway aspartyl proteases. In this study, we identified five putative pfam00026 aspartyl proteases encoded by the K. lactis genome. A set of selectable marker-free protease deletion mutants was constructed in the prototrophic K. lactis GG799 industrial expression strain background using a PCR-based dominant marker recycling method based on the Aspergillus nidulans acetamidase gene (amdS). Each mutant was assessed for its secretion of protease activity, its health and growth characteristics, and its ability to efficiently produce heterologous proteins. In particular, despite having a longer lag phase and slower growth compared with the other mutants, a Δyps1 mutant demonstrated marked improvement in both the yield and the quality of Gaussia princeps luciferase and the human chimeric interferon Hy3, two proteins that experienced significant proteolysis when secreted from the wild-type parent strain.
Asunto(s)
Proteasas de Ácido Aspártico/deficiencia , Expresión Génica , Kluyveromyces/enzimología , Kluyveromyces/metabolismo , Proteínas Recombinantes/metabolismo , Arecaceae/enzimología , ADN de Hongos/química , ADN de Hongos/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Kluyveromyces/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Análisis de Secuencia de ADNRESUMEN
Purifying proteins from recombinant sources is often difficult, time-consuming, and costly. We have recently instituted a series of improvements in our protein purification pipeline that allows much more accurate choice of expression host and conditions and purification protocols. The key elements are parallel cloning, small scale parallel expression and lysate preparation, and small scale parallel protein purification. Compared to analyzing expression data only, results from multiple small scale protein purifications predict success at scale-up with greatly improved reliability. Using these new procedures we purified eight of nine proteins from xenotropic murine leukemia virus-related virus (XMRV) on the first attempt at large scale.
Asunto(s)
Clonación Molecular/métodos , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/química , Animales , Baculoviridae/genética , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Spodoptera , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/genética , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/metabolismoRESUMEN
Griffithsin (GRFT) is a novel anti-HIV protein isolated from the red alga Griffithia sp. The potent anti-viral activity of GRFT against both laboratory and primary isolates of HIV at picomolar concentrations makes this protein an attractive candidate microbicide to prevent sexual transmission of HIV. Here, we describe the recombinant production and purification of a biologically active hexa-histidine-tagged GRFT (His-GRFT) from Escherichia coli. To facilitate a large-scale production of recombinant His-GRFT, we tested different expression conditions to optimize the expression in the cytoplasm of E. coli to increase the overall production of soluble His-GRFT. Attempts to express His-GRFT in shake flask cultures resulted in a modest yield of soluble His-GRFT, with a large accumulation of the protein in inclusion bodies. The use of a fermenter and of a rich, auto-inducing medium allowed the total amount of His-GRFT per liter to be increased by about 45-fold, with approximately 70% of the protein expressed in the soluble fraction. N-terminal sequencing and MALDI-TOF analyses of the recombinant His-GRFT confirmed that the initial methionine residue was cleaved off. Recombinant His-GRFT showed equivalent activity with natural GRFT, both in respect to gp120-binding characteristics as well as anti-HIV activity. Size-exclusion chromatography analysis showed that both native GRFT and recombinant His-GRFT existed as homodimers in solution. The expression system described in this work provides a basis for the mass production of GRFT to allow further studies of the protein and investigation of therapeutic and preventive strategies against HIV.
Asunto(s)
Proteínas Algáceas/genética , Reactores Biológicos , Infecciones por VIH/prevención & control , Lectinas/genética , Proteínas Recombinantes/genética , Rhodophyta/química , Rhodophyta/fisiología , Inactivación de Virus , Proteínas Algáceas/biosíntesis , Proteínas Algáceas/fisiología , Secuencia de Aminoácidos , Clonación Molecular/métodos , Lectinas/biosíntesis , Lectinas/fisiología , Datos de Secuencia Molecular , Lectinas de Plantas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , SolubilidadRESUMEN
Secretion of a recombinant protein from the yeast Pichia pastoris requires the presence of a signal peptide at the amino terminus. Maintaining the full amino acid sequence of the signal peptide is thought to be important for proper signal processing and protein secretion. We show that at least for one protein, a synthetic human interferon, the presence of a Gateway recombination site within the signal peptide is fully compatible with high levels of protein secretion. The amino termini of the secreted interferon proteins cloned with Gateway and cloned with restriction enzymes and ligase are identical, and the proteins were highly active in biological assays. Compatibility with Gateway cloning simplifies construction of plasmids directing secretion of recombinant proteins from P. pastoris.