Polymeric microspheres as protein transduction reagents

David Nagel, Jonathan M. Behrendt, Gwen F. Chimonides, Elizabeth E. Torr, Andrew Devitt, Andy J. Sutherland, Anna V. Hine

Research output: Contribution to journalArticle

Abstract

Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere-protein linkage was stable for ≥90 h at 37°C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake.

LanguageEnglish
Pages1543-1551
Number of pages9
JournalMolecular and Cellular Proteomics
Volume13
Issue number6
Early online date1 Apr 2014
DOIs
Publication statusPublished - 1 Jun 2014

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Microspheres
Proteins
Transfection
Human Immunodeficiency Virus Proteins
Endosomes
Functional analysis
Lysosomes
Liposomes
Nucleic Acids
Proteolysis

Bibliographical note

Creative Commons Attribution license (CC-BY). This work was funded by EPSRC

Grant No. EP/D038057/1 to A.V.H. and A.J.S.

This article contains supplemental material.

Cite this

Nagel, David ; Behrendt, Jonathan M. ; Chimonides, Gwen F. ; Torr, Elizabeth E. ; Devitt, Andrew ; Sutherland, Andy J. ; Hine, Anna V. / Polymeric microspheres as protein transduction reagents. In: Molecular and Cellular Proteomics . 2014 ; Vol. 13, No. 6. pp. 1543-1551.
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Polymeric microspheres as protein transduction reagents. / Nagel, David; Behrendt, Jonathan M.; Chimonides, Gwen F.; Torr, Elizabeth E.; Devitt, Andrew; Sutherland, Andy J.; Hine, Anna V.

In: Molecular and Cellular Proteomics , Vol. 13, No. 6, 01.06.2014, p. 1543-1551.

Research output: Contribution to journalArticle

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