Transient overexpression levels of the wild type and the E164K haboring Benchwarmer protein in HEK293 cell

Abstract

      Human Benchwarmer (BNCH) encodes a transmembrane helices protein belonging to the Major Facilitator Superfamily found in lysosomes. Genetic abrogations of BNCH caused neurodegeneration, accumulation of substances in lysosomes, shorter lifespan, and early onset of senescence in fruit flies, zebrafish, and roundworms. However, the molecular function of BNCH protein remains to be identified. To develop a cell assay to characterize the molecular function of the protein, we have cloned the coding sequences of the wild type BNCH and the BNCH carrying E164K loss of function mutation in the mammalian expression vector pcDNA 3.1 and expressed both vectors in the HEK293 cell line. The mutant E164 must be proved not to cause apparent changes in the expression levels and the location of the mutated BNCH thereby it can be used as a counter control in a cellular assay to assess the function of BNCH. In this study, we used lipoplexes to transfect and transiently express the WT BNCH and E164K BNCH in HEK293 cell line. The protein expression levels were evaluated by Western Blot by using the specific antibody. Our results show that both BNCH isoforms were successfully transfected and equally overexpressed in the HEK293 cells. Therefore, the cell lines will serve as important tool to investigate the molecular role of BCHN in the further study.