Extração, purificação e caracterização físico-química da proteína verde fluorescente recombinante (GFPuv) expressa em Escherichia coli

O aumento do uso da proteína verde fluorescente (GFPuv) como ferramenta de pesquisas biotecnológicas requer um estudo mais cuidadoso das propriedades bioquímicas e físicas da molécula de GFPuv. Este trabalho teve como objetivo a aplicação de métodos físicos e químicos para o isolamento, a extração da GFPuv de células de Escherichia coli DH5-±, purificação da proteína, e o estudo da estabilidade desta em diferentes valores de pH. Células de E. coli expressando GFPuv foram submetidas a quatro ciclos sucessivos de congelamento e descongelamento intercalados por sonicação (CDS), para promover a permeação seletiva da GFPuv. Os permeados foram submetidos à extração por partição em três fases (TPP) e posterior purificação por eluição da proteína em coluna cromatográfica de interação hidrofóbica (HIC).Obteve-se rendimento semelhante em GFPuv no 1º ciclo de permeação seletiva (CDS) e por extração (TPP) associada à purificação (HIC) para os quais impurezas não foram visualizadas por eletroforese. As estruturas moleculares da GFPuv extraída e purificada mostraram-se inalteradas em valores de pH entre 6,0 e 9,8, e foram confirmadas nos espectros de emissão e de excitação.The recombinant green fluorescent protein (GFPuv) has been used as a marker in several research fields. The purpose of the present work was to evaluate the influence of the selective physical permeation procedure applied to the cells of Escherichia coli for the extraction of GFPuv in relation to the chemical procedures of extraction and purification. Transformed cells (0.92-1.44 mg/mL) of E. coli DH5-a expressing GFPuv were submitted to four cycles (1º, 2º, 3º, 4º) of freezing (-20 ºC/ 0.83 ºC/ min/thawing interlaid by sonication (3 pulses/6 s/25 vibrations). The intracellular permeate with GFPuv in buffer solution (Tris-HCl 25 mM pH 8.0 + b-mercaptoethanol (1 mM) + PMSF (0.1 mM)) was submitted to the three-phase partitioning (TPP) method and subsequent purification through hydrophobic interaction chromatography column (HIC). The results showed that the first selective permeation cycle applied to the cells was more efficient in the release of the protein from the cell fragments without subsequent extraction and purification in relation to the second, third and fourth permeation procedures. The permeated, extracted and purified GFPuv showed similarity to the standard GFPuv characteristics of fluorescence, stability and solubility

Expression of green fluorescent protein (GFPuv) in Escherichia coli DH5-a, under different growth conditions

The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-α grown in LB/amp broth at 37oC, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by a fractional factorial (24-1) design at two levels: (i) the effect of storing (24-48 h) the seeded broth at 4oC prior to incubation at 37oC; (ii) the effect of agitation speed (100-200 rpm); (iii) the final concentration (0.05-0.5 mM) of IPTG (isopropyl–β-D-thiogalactopyranoside); and (iv) the addition of IPTG at set cell densities (OD660 0.01-0.8). GFPuv was extracted from cells by the three phase partitioning method (TPP) and further purified with a methyl HIC column. The cultures grown at 37oC/24 h provided the highest yields of GFPuv under the conditions: (i) pre-storage at 4oC/24 h; (ii) agitation speed at 100 rpm; (iii) 0.5 mM IPTG; (iv) IPTG addition at OD660=~0.01. On the other hand, at 37oC/ 8 h, GFPuv expression was dependent upon agitation of broth cultures at 200 rpm and the IPTG addition at the beginning of the growth exponential phase. Key Words: Green fluorescent protein (GFPuv), Escherichia coli DH5-α, growth kinetic parameters, expressed GFPuv kinetic parameters, three phase partitioning extraction (TPP). African Journal of Biotechnology Vol.3(1) 2004: 105-11

Tratamento químico de hortaliças poluídas

The effectiveness of chemicals as desinfectants for reducing the parasite contamination of lettuce leaves (Lactuca sativa, L.) was investigated. Potassium permanganate at 1: 5000 and 1: 10000 was employed, while sodium hypochlorite compound was used in con centrations of 10, 15, 20, 25, 30 and 40 ppm. In all experiments the contact time was 5,10, 20 and 40 minutes. The expousure of lettuce leaves to sodium hypochlorite 40 ppm of free cloro for 10 minutes was found to be highly effective.Das amostras de hortaliças em folhas coletadas no período de abril a agosto de 1981, no Entreposto Comercial da Cidade de São Paulo, SP (Brasil), 75% e 57% apresentaram contagens de coliformes fecais e de Escherichia coli, por grama de produto, superiores a 2 x 10² respectivamente; e em 85% constatou-se a presença de parasitas protozoários ou/e helmintos. O tratamento químico das folhas de alface (Lactuca sativa), com solução de hipoclorito de sódio, por um período de exposição de 10 min à concentração de 40 ppm de cloro livre, mostrou-se eficaz do ponto de vista parasitológico

Stability of furosemide and aminophylline in parenteral solutions

Parenteral solutions (PS) are one of the most commonly used drug delivery vehicles. Interactions among the drug, components in the drug's formulation, and the PS can result in the formation of inactive complexes that limit efficacy or increase side effects. The aim of this work was to evaluate possible interactions between the drugs and PS, assess drug stability and to identify degradation products after 20 h at room temperature. Furosemide (FSM) and Aminophylline (APN) were added to PS containing either 20% mannitol or 0.9% NaCl at pH 6.5-7.5 and 10-11. Their behavior was studied individually and as an admixture, after 1 h oxidation with H2O2, using a spectrophotometer and HPLC. Individually, FSM and APN added to 20% mannitol and 0.9% NaCl solutions had the highest stability at pH 10-11. When FSM and APN were combined, the behavior of FSM was similar to the behavior observed for the drug individually in the same solutions. With the drugs combined in 20% mannitol pH 10-11, HPLC showed that both drugs were stable after a 20 h period yielding two distinct peaks; in oxidized samples, the elution profile showed four peaks with retention times unrelated to the untreated samples.Soluções parenterais de grande volume são frequentemente utilizadas no ambiente hospitalar para a veiculação de fármacos. No entanto, possíveis incompatibilidades entre as estruturas dos fármacos, em diferentes veículos de administração, podem gerar possíveis associações antagônicas ou sinérgicas, resultando em alterações das propriedades físico-químicas, consequentemente, dos efeitos farmacológicos e das respostas clínicas esperadas. Este artigo avaliou a estabilidade e a possível formação de produtos de degradação entre os fármacos furosemida e aminofilina quando estes foram veiculados em soluções parenterais, após o preparo e após o período de 20 h. Furosemida e aminofilina foram adicionadas às soluções de 20% manitol e 0,9% NaCl nos valores de pH 6,5-7,5 e 10-11. A estabilidade dos fármacos foi avaliada individualmente, combinada e após degradação com peróxido de hidrogênio através de espectrofotometria de UV e HPLC. Furosemida e aminofilina individualmente avaliadas mostraram alta estabilidade em ambas as soluções estudadas nos valores de pH 10-11. Quando os fármacos foram combinados o comportamento da furosemida foi similar ao observado na ausência de aminofilina. Os fármacos combinados em 20% manitol pH10-11 por HPLC foram estáveis após o período de 20 h. Após degradação o perfil de cromatograma encontrado foi diferente do observado na ausência de degradação mostrando que o método é indicativo de estabilidade.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Pesquisa (CNPq)Coordenadoria de Apoio a Pesquisa (CAPES

Methods of endotoxin removal from biological preparations : a review

ABSTRACTPURPOSE: Endotoxins, also called lipopolysaccharides (LPS), are major contaminants found in commercially available proteins or biologically active substances, which often complicate study of the biological effects of the main ingredient. The presence of small amounts of endotoxin in recombinant protein preparations can cause side effects in host organism such as endotoxin shock, tissue injury, and even death. Due to these reactions, it is essential to remove endotoxins from drugs, injectables, and other biological and pharmaceutical products. An overview of this subject is provided by this article. METHODS: An extensive review of literature with regard to methods for removal of endotoxin from biotechnological preparations was carried out. RESULTS: A short history of endotoxin is presented first. This is followed by a review of chemical and physical properties of endotoxin and its pathophysiological effects when the body is exposed to LPS excessively or systemically. The techniques of endotoxin determination and interaction of endotoxin with proteins is also presented, taking into consideration the established techniques as well as the state of the art technology in this field. A review of techniques of endotoxin mentioned with relatively high protein recoveries; however, special attention is given to two-phase aqueous micellar systems, which are valuable tools for endotoxin removal from pharmaceutical proteins on a small scale because they provide a mild environment for biological materials. CONCLUSIONS: Efficient and cost-effective removal of endotoxins from pharmaceutical and biotechnology preparations is challenging. Despite development of novel methods, such as the two- phase aqueous micellar systems, in recent years, more research is needed in this field

Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system

BACKGROUND: A typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments. METHODOLOGY: Samples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method. RESULTS: The 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%. CONCLUSIONS: We found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments

Bacillus atrophaeus inactivated spores as a potential adjuvant for veterinary rabies vaccine

Rabies is a viral encephalitis, nearly always fatal, but preventable through vaccines. Rabid animal bite is the prime transmission act, while veterinary vaccination is one of the best strategies for rabies general prevention. Aluminum compounds and saponin are the commercial adjuvants used for this vaccine nowadays. Nevertheless, aluminum compounds can provoke undesired side effects and saponin has a narrow activity range without toxicity. B. atrophaeus inactivated spores (BAIS), with or without saponin, were then used as an alternative to boost the inactivated rabies virus response. BAIS was as effective as saponin in augmenting antibody titers, but combination of both adjuvants doubled the titers raised by them individually. The combined adjuvant formulation maintained viability for 21 months when stored at 4-8 degrees C. Overall, BAIS was demonstrated as a viable alternative to commercial adjuvants, while its combination with saponin resulted in even higher vaccine potency with good stability. (C) 2012 Elsevier Ltd. All rights reserved.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Butantan InstituteButantan Institut