Thermostable exoshells fold and stabilize recombinant proteins

Siddharth Deshpande,Goutam Chakraborty,Nihar D Masurkar,Malan Desai,Vallerinteavide Mavelli Girish,Juliana M Chan,Chester L Drum

doi:10.1038/s41467-017-01585-2

Siddharth Deshpande, Goutam Chakraborty + Show 5 more

Open Access

https://doi.org/10.1038/s41467-017-01585-2

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Abstract

The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.

Highlights

The expression and stabilization of recombinant proteins is fundamental to basic and applied biology
These C-termini have a molecular mass of ~ 21 kDa, which would likely interfere with the internalized protein folding and function
The pathway from a nascent polypeptide to a functional protein structure is determined by a balance of factors, some working in favor of, and many against, the successful folding of the end product[21, 22]

Summary

Introduction

The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. To improve recombinant protein expression and product stabilization using a single technology, we have engineered the AfFtn assembly derived from A. fulgidus, to create a thermostable exoshell (tES). To demonstrate the effect of tES on recombinant Escherichia coli expression and in vitro folding, we prepared genetic fusions (tESPOI) between a histidine-tagged tES monomer and three divergent POI (Supplementary Fig. 1c).

Results

Conclusion