Soluble T-cell receptor design influences functional yield in an E. coli chaperone-assisted expression system.

Kristin Støen Gunnarsen,Nicolay Rustad Nilssen,Kaare Bjerregaard-Andersen,Geir Åge Løset,Inger Sandlie,Ludvig Magne Sollid,Ralf Stefan Neumann,Lene Støkken Høydahl,Marek Cebecauer

doi:10.1371/journal.pone.0195868

Kristin Støen Gunnarsen, Nicolay Rustad Nilssen + Show 7 more

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https://doi.org/10.1371/journal.pone.0195868

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Abstract

There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads.

Highlights

The T-cell receptor (TCR) plays a central role in adaptive immunity by mediating recognition of peptides presented by the major histocompatibility complex (MHC) on the surface of antigen presenting cells
The single-chain TCR (scTCR) construct was generated by connecting the variable domains of the TCR with a flexible linker in a Vα-linker-Vβ orientation which was found to be the preferred orientation in a previous study [20]
In previous studies we have reported that over-expression of the chaperone FkpA has a major effect on soluble periplasmic expression of scTCRs, display levels on phage as well as selection performance of phage libraries [17, 20, 26]

Summary

Introduction

The T-cell receptor (TCR) plays a central role in adaptive immunity by mediating recognition of peptides presented by the major histocompatibility complex (MHC) on the surface of antigen presenting cells. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. GÅL is in part employed by the commercial company Nextera AS. The funder provided support in the form of salary for author GÅL, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ’author contributions’ section

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