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Clonagem e expressão de proteínas de ligação à penicilina e pseudomonas aeruginosa
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Abstract(s)
A Pseudomonas aeruginosa é uma bactéria Gram-negativa metabolicamente versátil e oportunista
que pode causar diversas infeções graves e persistentes em doentes com graves condições
médicas. O tratamento destas infeções é dificultado pela resistência adaptativa da bactéria, que
apresenta capacidade para crescer formando biofilmes e produzir uma variedade de fatores de
virulência. Estes fatores de virulência são importantes para neutralizar as defesas do hospedeiro e
provocar danos nos tecidos do mesmo. O tratamento de infeções causadas por esta bactéria é
dificultado pela elevada resistência a diversos antibióticos.
As proteínas de ligação a penicilinas (PBPs) são enzimas essenciais na síntese dos peptidoglicanos
presentes na parede celular. Os peptidoglicanos permitem que as bactérias resistam a pressões
elevadas, dão forma à célula bacteriana, e participam na divisão celular, entre outras funções. Ao
longo dos anos, estas enzimas têm sido alvo de investigação, pois estão envolvidas nos
mecanismos de resistência a diversos antibióticos β-lactâmicos.
Neste trabalho, pretendeu-se clonar os genes de P. aeruginosa que codificam para as PBPs mais
relevantes em termos clínicos em sistemas de expressão E.coli-pET28a(+). Posteriormente estas
proteínas serão utilizadas, no grupo de investigação, para o desenvolvimento de novos fármacos.
As proteínas selecionadas para a clonagem foram a PBP1b, PBP4 e PBP7. Inicialmente, extraiu-se
o gADN de P. aeruginosa e amplificaram-se por PCR as regiões a clonar utilizando primers
específicos. Os produtos de amplificação obtidos apresentaram os tamanhos esperados.
Posteriormente, as sequências de interesse foram digeridas e inseridas em vetores de expressão
pET28a(+). Foram obtidos transformantes positivos apenas para a proteína PBP7. A enzima PBP7
foi selecionada para sobre-expressão e purificação por cromatografia de afinidade em matriz de
níquel. Confirmou-se a presença da proteína em todas as etapas por SDS-PAGE. Analisando os
resultados é possível verificar uma banda de interesse com um peso molecular de
aproximadamente 33 kDa após a indução da expressão da proteína. No entanto, após a etapa de
purificação a banda correspondente à proteína de interesse não é visível. Futuramente, será
necessário otimizar a expressão e purificação da enzima PBP7 para que seja possível a sua
utilização no grupo de investigação para testar novos fármacos
Pseudomonas aeruginosa is a Gram negative bacteria known for its metabolic versatility that can cause severe persistent infections in chronic patients. The management of these infections is hampered by adaptive resistance of the bacteria, which can grown forming biofilms, and also produces a variety of virulence factors. These virulence factors, important to neutralize the host’ defences, can cause severe tissue damage. Treating these infections is also a complicated task due to their resistance to many antibiotics. Penicillin-binding proteins (PBPs) are enzymes that catalyse the synthesis of peptidoglycan, an essential component of the cell wall. Peptidoglycans allow bacteria to resist osmotic pressure, help cell maintain their shape, and play a role in cell division, among others. Throughout the years, these enzymes have been intensively studied as they are involved in the resistance mechanisms of bacteria to various β-lactam antibiotics. The goal of this work was to clone the P. aeruginosa genes that code the clinically relevant PBPs into the pET28a(+) expression vectors, affording proteins that will be used in the research group for drug design. PBP1b, PBP4 and PBP7 were the selected proteins. P. aeruginosa genomic ADN was extracted, and the gene coding regions were PCR amplified using specific primers, affording amplification products with the expected sizes. The amplicons were extracted and purified, and digested with restriction enzymes for insertion in pET28a(+) vectors. Positive transformants were obtained only for the PBP7 protein, which was then selected for over-expression and purification by immobilized metal ion chromatography on a nickel column. The presence of the protein was confirmed by SDS-PAGE. Results indicated the presence of an intense band with a molecular weight in the region of 33 kDa, as expected, after IPTG induction. However, upon purification, the protein could not be observed. In the future, the expression and purification of the PBP7 protein must be optimized in order for its use as a model assay for drug development.
Pseudomonas aeruginosa is a Gram negative bacteria known for its metabolic versatility that can cause severe persistent infections in chronic patients. The management of these infections is hampered by adaptive resistance of the bacteria, which can grown forming biofilms, and also produces a variety of virulence factors. These virulence factors, important to neutralize the host’ defences, can cause severe tissue damage. Treating these infections is also a complicated task due to their resistance to many antibiotics. Penicillin-binding proteins (PBPs) are enzymes that catalyse the synthesis of peptidoglycan, an essential component of the cell wall. Peptidoglycans allow bacteria to resist osmotic pressure, help cell maintain their shape, and play a role in cell division, among others. Throughout the years, these enzymes have been intensively studied as they are involved in the resistance mechanisms of bacteria to various β-lactam antibiotics. The goal of this work was to clone the P. aeruginosa genes that code the clinically relevant PBPs into the pET28a(+) expression vectors, affording proteins that will be used in the research group for drug design. PBP1b, PBP4 and PBP7 were the selected proteins. P. aeruginosa genomic ADN was extracted, and the gene coding regions were PCR amplified using specific primers, affording amplification products with the expected sizes. The amplicons were extracted and purified, and digested with restriction enzymes for insertion in pET28a(+) vectors. Positive transformants were obtained only for the PBP7 protein, which was then selected for over-expression and purification by immobilized metal ion chromatography on a nickel column. The presence of the protein was confirmed by SDS-PAGE. Results indicated the presence of an intense band with a molecular weight in the region of 33 kDa, as expected, after IPTG induction. However, upon purification, the protein could not be observed. In the future, the expression and purification of the PBP7 protein must be optimized in order for its use as a model assay for drug development.
Description
Keywords
Pseudomonas aeruginosa Proteínas de ligação à penicilina Clonagem Proteínas recombinantes Penicillin binding proteins Cloning Recombinant proteins