Search

La encapsulación de enzimas dentro de cápsides tipo virus llamadas “virus-like particles” (VLPs) es un tema de rápido crecimiento por su potencial uso como sistemas para suministro controlado de enzimas. Se ha determinado que ciertos tipos de cáncer presentan resistencia a los tratamientos de quimioterapia debido a una baja actividad de la enzima citocromo P450 en las células tumorales, es por ello que, en el presente trabajo, como estrategia para aumentar la eficiencia de activación de profármacos, se diseñaron nanopartículas virales del bacteriófago P22 con actividad citocromo P450 con NADPH como fuente de electrones. La metodología consistió en desarrollar dos sistemas de expresión para las dos variantes de la enzima citocromo P450 de Bacillus megaterium (CYPBM3) (WMV y TFFIS), cada una de las cuales esta fusionada al extremo carboxilo terminal de la proteína de andamiaje del bacteriófago P22, utilizando pBAD como vector de expresión. Asimismo, se utilizó el vector pRSF que contiene el gen que codifica para la proteína de la cápside viral,bajo el control del promotor lac. Ambos vectores de expresión se introdujeron a E. coli BL21 (DE3) y posteriormente se realizó la expresión heteróloga de ambas proteínas de manera secuencial para La encapsulación De la enzima CYPBM3 dentro de la cápside del bacteriófago P22. Las cápsides virales formadas fueron purificadas mediante ultracentrifugación y posteriormente por FPLC dando lugar a nanobiorreactores con alto grado de pureza, monodispersos en tamaño y catalíticamente activos utilizando NADPH como fuente de electrones. Estas partículas podrán ser funcionalizadas para ser reconocidas e internalizadas por células cancerosas. El aumento de la actividad CYPBM3 incrementará la eficiencia de los tratamientos de quimioterapia reduciendo las dosis y por ende los efectos secundarios. The encapsulation of enzymes within virus capsids called “Virus like particles (VLPs)” is a fast-growing Research field due to their potential use as carriers for the controlled supply of enzymes. It is well known that certain types of cáncer show resistance to chemotherapy treatments due to alow activity of the cytochrome P450 enzyme in tumor cells. In the present work, as a strategy to increase the efficiency of prodrugs activation, P22 bacteriophage viral nanoparticles containing cytochrome P450 activity with NADPH as a source of electrons have been developed. The methodology consisted in the design of two expression systems for Bacillus megaterium cytochrome P450 variants (CYPBM3 WMV and TFFIS), which are fused to a carboxi-terminal fragment of the scaffolding protein from P22 bacteriophage, using pBAD expression system. On the other hand, P22 capsid-encoded gene was cloned into pRSF vector, under the lac promoter control. Subsequently, the heterologous sequential expression of both proteins was performed in E. coli BL21 (DE3). The viral capsids were purified by ultracentrifugation and FPLC generating nanobioreactors with high degree of purity, monodispersed and catalytically active using NADPH as a source of electrons. These particles could be functionalized to be recognized and internalized by cáncer cells. The increase of the activity CYPBM3 will increase the efficiency of chemotherapy treatments, thus reducing doses and concomitantly, side effects.

Computer simulations are used in situations where the object of study can not be investigated by traditional methods, or not to use more of the test organisms that have already been used. Simulations are useful to test hypotheses or as a support tool to observing other results. The agent based modeling is a powerful and flexible tool that supports simulation experiments in the laboratory, biologists are being used in conjunction with computer specialists as a valuable tool to investigate the properties of biological systems. In this paper, an agent-based model is presented, from a bio-marker enzyme experiment run at the laboratory with mussel Mytilus edulis, marine species of the coast of Baja California, Mexico, exposed to oil dispersant (Nokomis 3-F4) obtained in toxicity tests, also Net Logo simulation tool is used to show the impact over three different tissues (enzymatic activity) of the mussel produced by the oil dispersant.