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Cloning human Benchwarmer gene (BNCH) harboring E164K in vector pcDNA3.1 by site-directed mutagenesis method / Nguyen Hoang Danh, Vu Minh Thiet // Tạp chí Khoa học và Công nghệ - Đại học Nguyễn Tất Thành . - 2020. - tr. 06-11. - ISSN: 2615-9015
Ho Chi Minh city : Nguyen Tat Thanh University, 2020
6 p.
Ký hiệu phân loại (DDC): 660.6
Benchwarmer (BNCH) gene encodes an orphan transmembrane transporter belonging to the Major Facilitator Superfamily (MFS), facilitating the transport of ions, amino acids, simple sugars and recently lysolipids. The loss of BNCH function caused lethality in several animal models with neurodegeneration and senescence. At the cellular level, dysregulation of BNCH leads to adverse phenotypes of lysosome and also autophagy (i.e. dyshomeostasis, accumulation of carbohydrates and sphingolipids, and enlarged lysosome). However, the molecular function and ligand of BNCH protein remain to be unrevealed. This study aims to create a radical substitution change in human BNCH coding gene to knock out the protein functions. More specifically, lysine (K) was used to replace the glutamic acid residue 164 (E164K) which is conserved in many animals (fly, zebrafish, mouse and human) and this E164K mutation recapitulated BNCH mutant phenotype. In conclusion, BNCH harboring E164K (BNCH*) was successfully produced by site-directed mutagenesis and cloned into pcDNA.3.1 vector. The construct was transformed into E. coli OmniMAX and that provides a valuable cell assay to search for the molecular ligand of BNCH.
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