Efficient Mutagenesis, Expression and Purification of Procathepsin D
High level expression of proteins in mammalian cells and the ability to distinguish an expressed protein from its endogenous counterpart are necessary when examining the structure/function relationships of many ubiquitously expressed proteins, such as the lysosomal aspartyl protease procathepsin D. We have achieved a high level of protein expression by transient transfection into 293 cells (a human embryonal kidney cell line transformed with adenovirus). Distinction between endogenous and introduced protein has been accomplished by fusing a 13 amino acid segment derived from the c-myc sequence to the C-terminus of procathepsin D. This fusion was devised by H.R.B. Pelham (1988). The myc extension permits the isolation and purification of the expressed protein by immunoaffinity chromatography with an available monoclonal antibody, 9E10, directed against 11 amino acids of the peptide. Investigation of structure/function relationships requires repeated rounds of mutagenesis in vitro followed by expression and assay in mammalian cells. We have facilitated this process by constructing a vector that can be shuttled between bacterial and mammalian hosts without any intermediate subclonings.
KeywordsConditioned Medium Immunoaffinity Chromatography Human Embryonal Kidney Cell Line Amino Acid Segment Carboxyl Protease
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