The DOI matches its Crossref record; 45 of the 45 references it cites were found in a registry or an index.
Soluble T-cell receptor design influences functional yield in an E. coli chaperone-assisted expression system
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.
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Self-citation
Research works printed but not linked: 1
- GA Løset, 2007, Functional phage display of two murine alpha/beta T-cell receptors is strongly dependent on fusion format, mode and periplasmic folding assistance, Protein engineering, design & selection: PEDS, 20, 461, 10.1093/protein/gzm044 · DOI
Metadata
- Authors
- Gunnarsen, K. S., Høydahl, L. S., Neumann, R. S., Bjerregaard-Andersen, K., Nilssen, N. R., Sollid, L. M., Sandlie, I., & Løset, G. Å.
- Venue
- PLOS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2018-04-12
- Volume
- 13
- Issue
- 4
- Pages
- e0195868
- ISSN
- 1932-6203
- Cited by
- 15
- References
- 45
- Language
- en
- Funders
- South-Eastern Norway Regional Health Authority (grant 2012046)
- Research Council of Norway (grant 197085)
- Research Council of Norway through its Centers of Excellence funding scheme (project number 179573/V40)
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