RESUMEN
Pseudomonas aeruginosa exotoxin A (PE) is a bacterial toxin composed of three domains namely: cell binding, translocation and enzymatic domain. The cytotoxic activity of PE is attributed to the enzymatic domain, which inhibits protein synthesis through ADP-ribosylation of EF-2. PE can be genetically modified to fight cancer. In this regard, a truncated and modified form of PE was produced that could be used for more potent immunotoxins. This modified form termed PE38KDEL was completely devoid of cell binding domain and parts of translocation domain II and Ib which are reported to be inessential for cytotoxicity of the toxin. The resultant expressed protein consisted of the essential translocation domain II and catalytic subunit (domain Ib, III). The deletions in the exotoxin A gene for truncated protein production were made via overlapping PCR extension. The amplicon was cloned in pTZ57r-T vector for DNA works and sub cloned in pET22b expression vector. It is demonstrated here that PE38KDEL can be expressed in huge quantities in Escherichia coli by using the recombinant vector PE38KDEL/pET under control of T7 promoter and E. coli host strain BL21 (DE3) CodonPlus. The protein expression was optimized at 0.5 mM IPTG concentration for induction as soon as the OD600 nm reached 0.6 with 6 hours of post induction culturing at 37°C. The recombinant protein was expressed both as soluble and inclusion body forms however the expression of the soluble form was more pronounced.
Asunto(s)
Toxinas Bacterianas/genética , Exotoxinas/genética , Pseudomonas aeruginosa/genética , Toxinas Bacterianas/metabolismo , Clonación Molecular , Escherichia coli/genética , Exotoxinas/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de SecuenciaRESUMEN
The current study was carried out to evaluate the effects of gamma irradiation on the epiphytic microflora and ripening process of the green Dwarf Cavendish bananas harvested at the three-quarter stage of the maturity. The mature green bananas were irradiated using Cobalt-60 as the source of irradiation at different dosages of 0.5, 0.75 and 1.0 kGy. The mean life of both the experimental and control group of fruits was analyzed under ambient conditions. For all the treatments the microbial potential, the decay percent and the ripening behavior of the fruits were recorded. Results revealed that the applied radiation doses reduced the decay incidence, delayed ripening process and greatly inhibit the microbial growth (total bacterial and fungal count) thereby enhancing the shelf life of bananas. Irradiation dose of 1.0 kGy was found to be the most effective dose to positively maintain the stored bananas under ambient conditions. The mean life of bananas was extended by 14 days. The identification of the enteric bacteriaeaceae through API 20 E strips revealed the presence of Shigella sonnie on the fruit surface along with Escherichia coli and a nonfermentor spp. The dominant spoilage causing fungi identified were Aspergillus niger, Aspergillus flavus, Collotrichum musae, Fusarium oxysporum,Mucor spp, Lasiodiplodia theobromea and Rhizopus stolonifer.
O Presente estudo foi realizado para investigar os efeitos da radiação gama sobre a microflora epífita e amadurecimento das bananas Cavendish Anão verde colhidas no estádio de três quartos da maturidade. As bananas verdes maduros foram irradiadas usando Cobalto-60 como fonte de irradiação a diferentes dosagens de 0,5, 0,75 e 1,0 kGy. A vida média de ambos os grupos experimental e de controlo de frutas foi analisada sob as condições ambientes. Para todos os tratamentos a potenciais microbiana, o percentual decadência e do comportamento do amadurecimento dos frutos foram recorded.Results revelou que as doses de radiação aplicadas reduziu a incidência de podridões, atrasou processo de amadurecimento e inibir significativamente o crescimento microbiano (contagem de bactérias e fungos total), assim aumentar a vida de prateleira das bananas. dose de irradiação de 1,0 kGy foi encontrada como sendo a dose mais eficaz para manter positivamente as bananas armazenada sob condições ambientes. A vida média de bananas foi prorrogado por 14 dias. A identificação do bacteriaeaceae entérico através de API 20 E tiras revelou a presença de Shigella sonnie sobre a superfície do fruto, juntamente com Escherichia coli e um nonfermentor spp. A deterioração dominante causando fungos identificados foram Aspergillus niger, Aspergillus flavus, Collotrichum musae, Fusarium oxysporum, Mucor spp, Lasiodiplodia theobromea e Rhizopus stolonifer.