Ponente
Descripción
Human neutrophil elastase (HNE) is a serine protease that displays great interest for its key rol in several pathologies, like chronic lung diseases and cancer. Studies that involved HNE as a therapeutic target for inhibitory substances require elevated quantities of the enzyme. HNE is commercialized in the market at very high prices and this represents an obstacle for our investigation. The objective of this work is to express recombinant elastase in human embrionary kidney cells 293T. A synthetic gene that codes for HNE was first cloned into a mammalian cell vector that expresses a six-histidine tag on the C-terminal of the protein. HEK 293T cells were transfected by polyethyleneimine method. Low quantities of the enzyme were detected only by ELISA in HEK 293T cells supernatant after transient transfection. A second cloning strategy was implemented by inserting the synthetic gene into a mammalian cell vector that expresses a human immunoglobulin G heavy chain fragment on the C-terminal of the protein. The success of cloning was confirmed by DNA sequence analysis. The transfection of HEK 293T cells by the same method is the next step for the future.
