Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

Robert B Quast,Andrei Sonnabend,Marlitt Stech,Stefan Kubick,Stefan M Schiller,Doreen A Wüstenhagen,Biljana Ballion,Péter Kele,Balázs R Varga

doi:10.1038/srep34048

Abstract

Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system.

Highlights

Orthogonal translation systems facilitating the incorporation of non-canonical amino acids have gained much attention over the last decades, providing researchers with efficient tools to implement desired characteristics into proteins in a site-directed manner[8,9,10,11]
The successful synthesis of the epidermal growth factor receptor (EGFR)-eYFP fusion protein was verified by different means (Fig. 1a–c), ligand-independent receptor activation based on autophosphorylation of Y1068 was not detectable
Fluorescent spheres in the confocal image of the microsomal fraction taken under hypo-osmotic conditions after four consecutive cell-free reactions reflected the EGFR-eYFP fusion protein to be localized at the microsomal membranes, thereby supporting the hypothesis of membrane embedment due to a directed translocation mediated by the N-terminal melittin signal peptide in the cell-free environment (Fig. 1d)

Summary

Introduction

Orthogonal translation systems facilitating the incorporation of non-canonical amino acids have gained much attention over the last decades, providing researchers with efficient tools to implement desired characteristics into proteins in a site-directed manner[8,9,10,11]. AzF is known to selectively react under physiological conditions via a set of different reactions such as Staudinger ligation, copper-catalyzed alkyne-azide cycloadditions and copper-independent strain-promoted alkyne-azide cycloadditions[14] In this context, bioorthogonal chemistry facilitates functional analysis of membrane proteins. The ability of an orthogonal cell-free translation system (OcfTS) composed of a mutant E. coli tyrosyl-tRNA synthetase and a natural amber suppressor tRNACUA to incorporate the non-canonical amino acid p-azido-L-phenylalanine (AzF) into cell-free synthesized EGFR at different positions in a site-directed manner and with high fidelity is demonstrated. The generation of covalently linked synthetic EGFR dimers is facilitated by a novel homobifunctional COMBO linker system

Methods

Results

Conclusion