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

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

doi:10.1371/journal.pone.0195868

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

PLoS One. 2018 Apr 12;13(4):e0195868. doi: 10.1371/journal.pone.0195868. eCollection 2018.

Authors

Kristin Støen Gunnarsen^{1

2

3}, Lene Støkken Høydahl^{1

2

3}, Ralf Stefan Neumann^{1

2}, Kaare Bjerregaard-Andersen⁴, Nicolay Rustad Nilssen^{1

2

3}, Ludvig Magne Sollid^{1

2

5}, Inger Sandlie^{1

2

3}, Geir Åge Løset^{1

2

3

6}

Affiliations

¹ Centre for Immune Regulation, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway.
² Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway.
³ Department of Biosciences, University of Oslo, Oslo, Norway.
⁴ Department of Chemistry, University of Oslo, Oslo, Norway.
⁵ KG Jebsen Coeliac Disease Research Centre and Department of Immunology, University of Oslo, Oslo, Norway.
⁶ Nextera AS, Oslo, Norway.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Escherichia coli / genetics*
Escherichia coli / metabolism
Gene Expression*
Models, Molecular*
Molecular Chaperones / metabolism
Protein Binding
Protein Conformation
Protein Multimerization
Receptors, Antigen, T-Cell / chemistry*
Receptors, Antigen, T-Cell / genetics*
Receptors, Antigen, T-Cell / isolation & purification
Receptors, Antigen, T-Cell / metabolism
Solubility
Structure-Activity Relationship

Substances

Molecular Chaperones
Receptors, Antigen, T-Cell

Grants and funding

The study was funded with grants from South-Eastern Norway Regional Health Authority (grant 2012046, https://www.helse-sorost.no/, KSG), the Research Council of Norway (grant number 197085, https://www.forskningsradet.no/en/Home_page/1177315753906, LSH), and the Research Council of Norway through its Centers of Excellence funding scheme, project number 179573/V40, LSH and GÅL). 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.