Can the Cellular Internalization of Cargo Proteins Be Enhanced by Fusing a Tat Peptide in the Center of Proteins? A Fluorescence Study

Xiaochao Chen, Jing Chen, Rong Fu, Pingfan Rao, Richard Weller, Jeremy Bradshaw, Shutao Liu

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The aim of this study was to investigate whether the cellular uptake of cargo proteins can be enhanced by fusing a Tat peptide in the center of proteins; glutathione-S-transferase (GST)-Tat-green fluorescent protein (GFP) and GST-GFP-Tat proteins were first constructed and expressed. The cellular internalization of both proteins was then evaluated and compared in HeLa cells using fluorescent microscopy and flow cytometry, as well as the transdermal delivery in human skin 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 stratum corneum 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 cell 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.

Original languageEnglish
Pages (from-to)879-886
Number of pages8
JournalJournal of Pharmaceutical Sciences
Volume107
Issue number3
Early online date11 Nov 2017
DOIs
Publication statusPublished - 1 Mar 2018

Keywords

  • cell-penetrating peptide
  • cellular internalization
  • confocal microscopy
  • fluorescence microscopy
  • green fluorescent protein
  • Tat peptide
  • transdermal delivery

ASJC Scopus subject areas

  • Pharmaceutical Science

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