Recombinant Cell-Permeable Puromycin N -Acetyltransferase Confers Puromycin Resistance by Direct Protein Transduction

Abstract

Puromycin N-acetyltransferase (PAC) is an enzyme that catalyzes the acetylation of puromycin, an inhibitor of protein synthesis. The PAC gene is often co-transfected with genes of interest in the same vector to serve as a selective marker, conferring puromycin resistance to mammalian cells. Cellpenetrating peptides (CPPs), which are 5-30 amino acids in length, facilitate the translocation of functional cargoes across the cell membrane. Among these, the HIV-transactivator of transcription (TAT) sequence is widely applied for its cell-penetrating and protein-delivery capabilities. In this study, we investigated whether attachment of the TAT sequence to PAC (TAT-PAC) enables intracellular delivery of TAT-PAC protein into mammalian cells, thereby conferring puromycin resistance. A recombinant TAT-PAC protein was expressed in Escherichia coli and purified to homogeneity. The purified TAT-PAC protein retained enzymatic activity, with a specific activity of 197 nmol/min/mg. Intracellular delivery of TAT-PAC was confirmed using confocal microscopy and flow cytometry, employing an RFP (red fluorescent protein)-tagged TAT-PAC fusion protein. Treatment of HEK293 and SY5Y cells with TAT-PAC resulted in increased cell viability in the presence of puromycin, demonstrating its functionality as a selection marker. This study suggests the potential application of cell-permeable PAC protein for selection of co-delivered therapeutic or gene-editing proteins in mammalian cells, providing a promising alternative to traditional genetic selection methods.