Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology.

Stefano Maffini,Katharina Overlack,Amal Alex,Giuseppe Ciossani,Soumitra Polley,Philipp Selenko,Marchel Stuiver,Arsen Petrovic,Stephanie Voss,Andrea Musacchio,Sabine Wohlgemuth,Yaowen Wu,Valentina Piano,Beate Voss

doi:10.7554/elife.48287

Abstract

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

Highlights

The traditional divide between in vivo, in cellulo and in vitro approaches in modern molecular cell biology has progressively been overcome in recent years largely due to new conceptual and technical advancements
These results suggested that EP maybe broadly applicable to transduce defined amounts of recombinant proteins into cultured mammalian cells, with individual cells harboring uniform concentrations of exogenously delivered proteins (Figure 1—figure supplement 1C–D)
We studied KRAS signaling in live cells electroporated with GFP-tagged and Tide Fluor 3 (TF3)-labeled human KRAS (i.e. EGFP-TF3-KRAS, final size ~50 kDa), a conformational sensor for GTPase activity (COSGA) obtained by chemical labeling of recombinant protein

Summary

Introduction

The traditional divide between in vivo, in cellulo and in vitro approaches in modern molecular cell biology has progressively been overcome in recent years largely due to new conceptual and technical advancements. On the other hand, have generated tools for studying and manipulating biological pathways in creative new ways, with innovations that include, amongst others, genetic code expansion to introduce unnatural aminoacids with new functionalities into a protein of interest (Davis and Chin, 2012), the development of fluorescent dyes. Introduction of protein variants into cells has been achieved by transient transfection of DNA plasmids encoding these entities. Recipient cells typically express the desired proteins at levels that vary between cell lines and individual cells (Kim and Eberwine, 2010). Stable transfection of cultured mammalian cells offers more uniform protein expression but the generation of stable clones is laborintensive and time-consuming (Kim and Eberwine, 2010)

Methods

Results

Conclusion