Aim to investigate whether the cellular uptake of cargo proteins can be enhanced by fusing a Tat peptide in the center of proteins, GST-Tat-GFP and GST-GFP-Tat proteins were firstly constructed and expressed. The cellular internalization of both proteins was then evaluated and compared in HeLa cells by using fluorescent microscopy and flow cytometry, as well as the transdermal delivery in human skin by using confocal microscopy. Results from in-vitro cell experiments showed that GST-Tat-GFP protein efficiently internalized into HeLa cells when a Tat peptide was fused in the center of proteins, whereas its efficiency is lower than that of GST-GFP-Tat protein with a Tat peptide terminal fused. Ex-vivo transdermal delivery data also demonstrated that the lower efficiency of GST-Tat-GFP penetrating through human SC layer when compared with GST-GFP-Tat. Furthermore, both GST-GFP-Tat and GST-Tat-GFP presented a various degree of a mixture of cytoplasmic diffuse staining and punctate surface staining, and the pattern of distribution varied considerably in HeLa cells experiments depending on the concentration of protein used. Therefore, an improved mechanism for Tat-conjugated proteins was proposed, in which Tat-conjugated proteins were first associated with cell membrane, then accumulated on the cell surface, and finally internalized into cells by pore formation mechanism.
- Journal Article