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

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

Author information

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.

PMID:
29649333
PMCID:
PMC5897000
DOI:
10.1371/journal.pone.0195868
[Indexed for MEDLINE]
Free PMC Article

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