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1.
BMC Biotechnol ; 8: 50, 2008 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-18485214

RESUMO

BACKGROUND: Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein. RESULTS: Secretion level of the fusion protein produced in vitro in BHK cells was approximately 30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04-1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of approximately 150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (approximately 32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (approximately 3 h). In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested. CONCLUSION: Both the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.


Assuntos
Butirilcolinesterase/farmacocinética , Rim/enzimologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica/farmacocinética , Animais , Butirilcolinesterase/sangue , Butirilcolinesterase/genética , Linhagem Celular , Cricetinae , Cabras , Humanos , Taxa de Depuração Metabólica , Camundongos , Camundongos Transgênicos , Albumina Sérica/genética , Suínos
2.
Proc Natl Acad Sci U S A ; 104(34): 13603-8, 2007 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-17660298

RESUMO

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat beta-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.


Assuntos
Butirilcolinesterase/metabolismo , Leite/efeitos dos fármacos , Leite/enzimologia , Intoxicação por Organofosfatos , Animais , Animais Geneticamente Modificados , Butirilcolinesterase/genética , Butirilcolinesterase/isolamento & purificação , Butirilcolinesterase/farmacocinética , Metabolismo dos Carboidratos , Carboidratos/análise , Regulação Enzimológica da Expressão Gênica , Cabras , Cobaias , Humanos , Camundongos , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética
3.
Domest Anim Endocrinol ; 31(2): 173-85, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16274952

RESUMO

Relaxin is a peptide hormone produced by a wide variety of mammals. In the horse, the placenta is the major source of relaxin. Since pure equine relaxin is difficult to obtain to study its role in the pregnant mare, the objectives of this study were to produce recombinant equine prorelaxin and characterize its immunological and biological activity. First, an equine relaxin gene cassette was transfected into immortalized bovine mammary epithelial (MAC-T) cells. Second, immunological activity of media conditioned by transfected MAC-T cells was tested by Western blotting and quantified using a homologous equine radioimmunoassay. Finally, bioactivity of the conditioned media was tested using the human monocyte cell line, THP-1, which exhibits a rapid and dose-dependent increase in the accumulation of cAMP upon binding relaxin. The results showed that conditioned media, concentrated 5x, yielded 4.11 +/- 0.81 ng/ml recombinant equine prorelaxin. In addition, a 19 kDa immunoreactive band, corresponding to the expected size of equine prorelaxin, was visualized by SDS-PAGE. THP-1 cells incubated with conditioned media (5x) from transfected cells, in the presence of forskolin (1 microM) and isobutylmethylxanthine (50 microM), showed an increase in cAMP production over media from mock-transfected cells alone. In conclusion, recombinant equine prorelaxin secreted by MAC-T cells was both immunologically and biologically active. This study demonstrates the first attempt to produce recombinant equine prorelaxin, important for further study of the role of relaxin in the mare.


Assuntos
Cavalos/genética , Cavalos/metabolismo , Proteínas Recombinantes/biossíntese , Relaxina/biossíntese , Relaxina/genética , Animais , Bioensaio/veterinária , Western Blotting/veterinária , Bovinos , Linhagem Celular , Meios de Cultivo Condicionados , AMP Cíclico/metabolismo , DNA/química , DNA/genética , Feminino , Humanos , Mutagênese Insercional , Precursores de Proteínas/genética , Precursores de Proteínas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Relaxina/farmacologia , Relaxina/fisiologia , Transfecção
4.
BMC Biotechnol ; 5: 9, 2005 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-15823198

RESUMO

BACKGROUND: Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice. RESULTS: The GUM-GFP cassette was constructed and transgenic mice were generated in order to study the promoter's tissue specificity, the GFP kidney specific expression and its subcellular distribution. Tissues collected from three GUM-GFP transgenic mouse lines, and analyzed for the presence of GFP by Western blotting and fluorescence confirmed that the GUM promoter drove expression of GFP specifically in the kidney. More specifically, by using immuno-histochemistry analysis of kidney sections, we demonstrated that GFP expression was co-localized, with endogenous uromodulin protein, in the epithelial cells of the thick ascending limbs (TAL) of Henle's loop and the early distal convoluted tubule in the kidney. CONCLUSION: The goat uromodulin promoter is capable of driving recombinant protein expression in the kidney of transgenic mice. The goat promoter fragment cloned may be a useful tool in targeting proteins or oncogenes in the kidney of mammals.


Assuntos
Biotecnologia/métodos , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica , Técnicas Genéticas , Proteínas de Fluorescência Verde/metabolismo , Rim/embriologia , Rim/metabolismo , Mucoproteínas/genética , Regiões Promotoras Genéticas , Animais , Southern Blotting , Western Blotting , Clonagem Molecular , Genes Reporter , Cabras , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Túbulos Renais/embriologia , Túbulos Renais/metabolismo , Alça do Néfron/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , Modelos Genéticos , Mucoproteínas/metabolismo , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Fatores de Tempo , Distribuição Tecidual , Transgenes , Uromodulina
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