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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Puromycin N-acetyltransferase (PAC) confers puromycin resistance, commonly used as a selectable marker in gene transfer.
• Cell-penetrating peptides (CPPs), like the HIV-transactivator of transcription (TAT) sequence, facilitate intracellular delivery of cargo molecules.
• Traditional methods rely on genetic integration of selectable markers, which can be inefficient and time-consuming.

Purpose of the Study:

• To investigate the efficacy of a TAT-PAC fusion protein for intracellular delivery and puromycin resistance in mammalian cells.
• To explore a non-genetic approach for conferring puromycin resistance using cell-permeable proteins.
• To assess the potential of TAT-PAC as a selection marker for co-delivered therapeutic or gene-editing proteins.

Main Methods:

• Recombinant TAT-PAC protein was expressed in E. coli and purified.
• Enzymatic activity of purified TAT-PAC was determined.
• Intracellular delivery was visualized using RFP-tagged TAT-PAC and confirmed by confocal microscopy and flow cytometry.
• Cell viability assays were performed on HEK293 and SY5Y cells treated with TAT-PAC in the presence of puromycin.

Main Results:

• Purified TAT-PAC protein exhibited significant enzymatic activity (197 nmol/min/mg).
• Confocal microscopy and flow cytometry confirmed intracellular uptake of TAT-PAC.
• HEK293 and SY5Y cells treated with TAT-PAC showed enhanced survival in puromycin-containing media.
• TAT-PAC effectively functioned as a selectable marker in mammalian cells.

Conclusions:

• Cell-permeable TAT-PAC protein can be successfully delivered into mammalian cells.
• TAT-PAC confers puromycin resistance, demonstrating its potential as a non-genetic selection marker.
• This approach offers a promising alternative for selecting cells receiving co-delivered therapeutic or gene-editing proteins.