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Publication
Production and characterization of INTERLEUKIN-9 and its receptor
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Abstract(s)
O principal objetivo deste projeto foi proceder á descoberta da estrutura da Interleucina-9 e do seu respetivo recetor. Esta Interleucina mostrou uma ligação positiva com inflamações, asma e alergias. Por este motivo a descoberta das suas estruturas é essencial para o desenvolvimento de um inibidor eficaz.
Para atingir este objetivo, este projeto foi dividido em dois objetivos principais. O primeiro baseou-se na produção e purificação da Interleucina-9 e do seu recetor. No segundo objetivo, procedeu-se à caracterização biofísica e cristalização das proteínas produzidas.
Para alcançar o primeiro objetivo, procedeu-se à utilização de vários vetores de modo a obter construções finais com o fim de melhorar a produção e a solubilidades destas duas proteínas. Ambas as proteínas foram expressas em todas as estirpes testadas. No entanto, o principal problema que enfrentamos ao usar células de E. coli foi o facto de todas as proteínas expressas encontravam-se em corpos de inclusão. Resultado em proteínas insolúveis. Embora não tivéssemos conseguido o refold da proteína para uma proteína solúvel, os resultados obtidos foram bastante promissores. No futuro, seria necessário repetir mais técnicas de refold de proteínas ou construções de vetores diferentes.
Além da produção em E. coli, também executamos a produção destas proteínas em células Schneirder 2 (S2), sendo estas células de inseto. Obtivemos uma boa expressão de ambas as proteínas e estas encontravam-se na forma solúvel.
Com estas proteínas produzidas procedemos para a caracterização biofísica e cristalização destas. Primeiro foi medida a espectro de dicroísmo circular que nos mostrou que ambas as proteínas estavam devidamente bem enroladas. Após a confirmação do complexo da Interleucina-9 com o recetor, lemos a temperatura de fusão da Interleucina-9 sozinha e do complexo. Estas medições mostraram que o complexo é mais estável que o recetor da Interleucina-9 sozinho.
A prova final de interação entre estas duas proteínas foi medida através da afinidade entre estas por uma Termoforese em Microescala (MST). As curvas medidas evidenciaram uma ligação adequada entre estas proteínas.
Finalmente, procedeu-se à cristalização da Interleucina-9 sozinha e do complexo. Infelizmente não obtivemos nenhuma forma cristalina do complexo após 51 dias de controle continuo. Ao contrário da Interleucina-9, com esta proteína for possível detetar a presença de duas formas cristalinas após dois dias. Os nossos ensaios iniciais deram resultados, porém os nossos esforços futuros serão aplicados em maior profundidade na otimização das condições de cristalização de ambas as proteínas e do complexo.
The main purpose of this project was to discover the structure of the Interleukin-9 and its receptor. This Interleukin has shown a positive linkage with inflammation, asthma and allergies. For this reason, the discovery of these structures is very important for the development of an effective inhibitor. In order to achieve such a goal, this work attempts to accomplish two major objectives. First, the production and purification of Interleukin-9 and its receptor. Second, the biophysical characterization and crystallization of the proteins. For the first part, we used several vectors to conduct several constructs in order to help with the production and the solubility of these two proteins. Both proteins were expressed in all tested strains. However, the main problem that we faced using E. coli was the fact that all the expressed proteins were in inclusion bodies, resulting in an insoluble form protein form. Although we could not refold the proteins to a soluble form, our refolding trials showed promising results. In the future, we could try to refold these proteins with other techniques or different constructs. Additionally, we tried the production of Interleukin-9 and its receptor in insect cells, Schneider 2 (S2) cells. We obtained a good expression of both proteins with these cells and they were in a soluble form. With these proteins, we turned our efforts to the second objective, the biophysical characterization and crystallization of the proteins. First, we measured the Circular Dichroism Spectrum that showed us that both proteins were folded. Then, after the confirmation of the complex with the Interleukin-9 and the receptor, we analyzed the melting temperature of the Interleukin-9 alone and the complex. These measurements showed that the complex is more stable than the Interleukin-9 receptor alone. The final proof of interaction was direct measurement of the affinity between those two proteins by a MicroScale Thermophoresis (MST). The measured curves evinced a proper binding between these proteins. Finally, we proceeded to the crystallization of the Interleukin-9 alone and the complex. Unfortunately, we did not obtain any crystal form of complex after 51 days of continuous controlling. For the Interleukin-9 alone as opposed to the previously observed results with the complex, we could detect the presence of two crystal forms of IL-9 alone after two days. Our crystallization trials gave initial hits and our further efforts will be applied in greater depth to the optimization of crystallization conditions for complex and both proteins alone.
The main purpose of this project was to discover the structure of the Interleukin-9 and its receptor. This Interleukin has shown a positive linkage with inflammation, asthma and allergies. For this reason, the discovery of these structures is very important for the development of an effective inhibitor. In order to achieve such a goal, this work attempts to accomplish two major objectives. First, the production and purification of Interleukin-9 and its receptor. Second, the biophysical characterization and crystallization of the proteins. For the first part, we used several vectors to conduct several constructs in order to help with the production and the solubility of these two proteins. Both proteins were expressed in all tested strains. However, the main problem that we faced using E. coli was the fact that all the expressed proteins were in inclusion bodies, resulting in an insoluble form protein form. Although we could not refold the proteins to a soluble form, our refolding trials showed promising results. In the future, we could try to refold these proteins with other techniques or different constructs. Additionally, we tried the production of Interleukin-9 and its receptor in insect cells, Schneider 2 (S2) cells. We obtained a good expression of both proteins with these cells and they were in a soluble form. With these proteins, we turned our efforts to the second objective, the biophysical characterization and crystallization of the proteins. First, we measured the Circular Dichroism Spectrum that showed us that both proteins were folded. Then, after the confirmation of the complex with the Interleukin-9 and the receptor, we analyzed the melting temperature of the Interleukin-9 alone and the complex. These measurements showed that the complex is more stable than the Interleukin-9 receptor alone. The final proof of interaction was direct measurement of the affinity between those two proteins by a MicroScale Thermophoresis (MST). The measured curves evinced a proper binding between these proteins. Finally, we proceeded to the crystallization of the Interleukin-9 alone and the complex. Unfortunately, we did not obtain any crystal form of complex after 51 days of continuous controlling. For the Interleukin-9 alone as opposed to the previously observed results with the complex, we could detect the presence of two crystal forms of IL-9 alone after two days. Our crystallization trials gave initial hits and our further efforts will be applied in greater depth to the optimization of crystallization conditions for complex and both proteins alone.