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Abstract(s)
A necessidade cada vez mais frequente de DNA plasmídico (pDNA) para ser
usado em Terapia Génica e vacinas de DNA levou à procura de novos meios de
recuperação e purificação de plasmídeos que possam ser utilizados em larga escala. Os
Sistemas de Duas Fases Aquosas (SDFAs) têm sido testados para esse fim pois são uma
técnica fácil, simples e eficaz. Contudo, devido à sua baixa selectividade, têm sido
maioritariamente utilizados nos passos iniciais da purificação. Os sistemas Dextrano
(Dex)/Polietilenoglicol (PEG) são os melhores estudados mas apresentam a
desvantagem do Dextrano ser um polímero de elevado custo. O Hidroxipropilamido
(HPS) tem sido utilizado como substituto para o Dex, baixando o custo da operação
cerca de sete vezes, contudo nunca foi utilizado para a purificação de pDNA. Nos
SDFAs também se têm utilizado ligandos de afinidade para o pDNA, de modo a
aumentar a afinidade do pDNA para a fase do PEG, havendo assim uma maior
percentagem de recuperação do produto. Contudo, os ligandos normalmente utilizados
têm a desvantagem de serem caros, tendo anteriormente sido estudados alguns
antibióticos como potenciais ligandos de afinidade para o pDNA. Dos antibióticos
estudados, o Berenil revelou-se o mais promissor pois apresenta uma elevada constante
de afinidade para o pDNA.
Assim, o principal objectivo deste trabalho consistiu em testar sistemas
HPS/PEG para a recuperação de pDNA e na utilização do Berenil como ligando de
afinidade sendo para isso necessário ligá-lo ao PEG.
A partição do pDNA e do RNA em vários sistemas HPS/PEG foi estudada
através de electroforeses em gel de agarose e a determinação da concentração de pDNA
nas fases dos sistemas foi realizada por fluorescência e cromatografia de interacção
hidrofóbica. A influência de vários sais no comportamento do pDNA e do RNA foi
igualmente avaliada.
Assim, em sistemas com HPS e lisado, o pDNA precipita na interfase dos
sistemas e o RNA particiona maioritariamente para a fase superior. O pDNA puro em
sistemas com HPS particiona da fase superior para a fase inferior, com o aumento da
concentração de sal, verificando-se portanto a influência da elevada concentração de
sais presentes nas soluções de lise alcalina. Verifica-se que, com a substituição do Dex por HPS, o comportamento do
pDNA altera-se no SDFA. Para sistemas com Dex e pDNA puro, o pDNA particiona
para a fase inferior dos sistemas, independentemente da concentração de sal presente.
Contudo em sistemas com HPS, o pDNA tende a particionar para a fase superior dos
sistemas, passando para a fase inferior com o aumento da concentração de sal.
Todos os sais estudados influenciam de igual modo o comportamento do pDNA
puro nos sistemas, fazendo com que ele particione da fase superior para a fase inferior,
podendo estes ser usados para melhorar a separação entre o pDNA e os seus
contaminantes.
A ligação do Berenil ao PEG utilizando diferentes protocolos não foi
conseguida, tendo-se obtido, em todas as reacções realizadas, os reagentes de partida.
The need of larger amounts of plasmid DNA (pDNA) to be used in Gene Therapy and DNA vaccines has led to the search of new means of recovery and purification of pDNA that can be used in large scale. Aqueous Two-Phase Systems (ATPS) have been tested to achieve this end since they are an easy, simple and effective technique. However given their low selectivity they have been mainly used in the first purification steps. Dextran (Dex)/Polyethylene glycol (PEG) systems are the best studied systems but have the disadvantage of Dex being an expensive polymer. Hydroxypropyl starch (HPS) has been used as a substitute of Dex, lowering the overall cost but it was never used to purify pDNA. In ATPS pDNA affinity ligands have also been used to increase pDNA affinity towards the PEG phase, thus increasing product recovery yield. However due to the high cost of the ligands normally used, the affinity of several antibiotics to pDNA has been previously determined. From those, Berenil has demonstrated potential to be used as an affinity ligand given its high affinity constant towards pDNA. So the main goal of this work was to test HPS/PEG systems to recover plasmid DNA and to use Berenil as an affinity ligand for which is needed to attach it to PEG. pDNA and RNA partition in several HPS/PEG systems was studied through agarose gel electrophoresis and pDNA concentration was determined by fluorescence and hydrophobic interaction chromatography. The influence of several salts in pDNA and RNA behaviour was also assessed. In systems containing HPS and lysate, pDNA precipitates in the interface and RNA partition to the top phase. Pure pDNA in HPS systems partitions from the top to the bottom phase with the increase of salt concentration, thus indicating the influence of high salt concentration present in alkaline lysis solutions. Substituting Dex with HPS, pDNA behaviour changes in the ATPS. In Dex and pure pDNA systems, pDNA partitions to the bottom phase, independently of salt concentration present. However, in HPS systems, pDNA partitions to the top phase, and moves to the bottom phase with the increase of salt in the system. All the studied salts influence equally pure pDNA behavior in the systems, moving it from the top to the bottom phase. These salts can be used to better separate pDNA from their contaminants. The attachment of Berenil and PEG using different protocols was not achieved. In all the reactions prepared, start compounds were obtained separately.
The need of larger amounts of plasmid DNA (pDNA) to be used in Gene Therapy and DNA vaccines has led to the search of new means of recovery and purification of pDNA that can be used in large scale. Aqueous Two-Phase Systems (ATPS) have been tested to achieve this end since they are an easy, simple and effective technique. However given their low selectivity they have been mainly used in the first purification steps. Dextran (Dex)/Polyethylene glycol (PEG) systems are the best studied systems but have the disadvantage of Dex being an expensive polymer. Hydroxypropyl starch (HPS) has been used as a substitute of Dex, lowering the overall cost but it was never used to purify pDNA. In ATPS pDNA affinity ligands have also been used to increase pDNA affinity towards the PEG phase, thus increasing product recovery yield. However due to the high cost of the ligands normally used, the affinity of several antibiotics to pDNA has been previously determined. From those, Berenil has demonstrated potential to be used as an affinity ligand given its high affinity constant towards pDNA. So the main goal of this work was to test HPS/PEG systems to recover plasmid DNA and to use Berenil as an affinity ligand for which is needed to attach it to PEG. pDNA and RNA partition in several HPS/PEG systems was studied through agarose gel electrophoresis and pDNA concentration was determined by fluorescence and hydrophobic interaction chromatography. The influence of several salts in pDNA and RNA behaviour was also assessed. In systems containing HPS and lysate, pDNA precipitates in the interface and RNA partition to the top phase. Pure pDNA in HPS systems partitions from the top to the bottom phase with the increase of salt concentration, thus indicating the influence of high salt concentration present in alkaline lysis solutions. Substituting Dex with HPS, pDNA behaviour changes in the ATPS. In Dex and pure pDNA systems, pDNA partitions to the bottom phase, independently of salt concentration present. However, in HPS systems, pDNA partitions to the top phase, and moves to the bottom phase with the increase of salt in the system. All the studied salts influence equally pure pDNA behavior in the systems, moving it from the top to the bottom phase. These salts can be used to better separate pDNA from their contaminants. The attachment of Berenil and PEG using different protocols was not achieved. In all the reactions prepared, start compounds were obtained separately.
Description
Keywords
Terapia genética Plasmídeos Polietilenoglicol Hidroxipropilamido