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dc.contributor.authorLunde, Elin
dc.contributor.authorLøset, Geir Å
dc.contributor.authorBogen, Bjarne
dc.contributor.authorSandlie, Inger
dc.date.accessioned2015-10-09T02:13:53Z
dc.date.available2015-10-09T02:13:53Z
dc.date.issued2010
dc.identifier.citationBMC Biotechnology. 2010 Aug 24;10(1):61
dc.identifier.urihttp://hdl.handle.net/10852/46811
dc.description.abstractBackground Whereas T cell receptors (TCRs) detect peptide/major histocompatibility complexes (pMHCs) with exquisite specificity, there are challenges regarding their expression and use as soluble detection molecules due to molecular instability. We have investigated strategies for the production of TCR-immunoglobulin (Ig) fusion proteins. Two different TCRs that are characteristic of a mouse model for idiotype (Id) dependent immune regulation were engineered. They are structurally unrelated with different variable (V), diversity (D) and joining (J) segments, but each share one V gene segment, either Vα or Vβ, with the well characterized murine TCR, 2C. Results Several TCR-Ig formats were assessed. In one, the TCR V domains were fused to Ig constant (C) regions. In others, the complete extracellular part of the TCR was fused either to a complete Ig or an Ig Fc region. All molecules were initially poorly secreted from eukaryotic cells, but replacement of unfavourable amino acids in the V regions improved secretion, as did the introduction of a disulfide bridge between the TCR C domains and the removal of an unpaired cysteine. A screening strategy for selection of mutations that stabilize the actual fusion molecules was developed and used successfully. Molecules that included the complete heterodimeric TCR, with a stabilizing disulfide bridge, were correctly folded as they bound TCR-specific antibodies (Abs) and detected pMHC on cells after specific peptide loading. Conclusions We show that fully functional TCR-Ig fusion proteins can be made in good yields following stabilizing engineering of TCR V and C region genes. This is important since TCR-Ig fusions will be important probes for the presence of specific pMHCs in vitro and in vivo. In the absence of further affinity maturation, the reagents will be very useful for the detection of kinetic stability of complexes of peptide and MHC.
dc.language.isoeng
dc.rightsLunde et al; licensee BioMed Central Ltd.
dc.rightsAttribution 2.0 Generic
dc.rights.urihttp://creativecommons.org/licenses/by/2.0/
dc.titleStabilizing mutations increase secretion of functional soluble TCR-Ig fusion proteins
dc.typeJournal article
dc.date.updated2015-10-09T02:13:54Z
dc.creator.authorLunde, Elin
dc.creator.authorLøset, Geir Å
dc.creator.authorBogen, Bjarne
dc.creator.authorSandlie, Inger
dc.identifier.cristin520758
dc.identifier.doihttp://dx.doi.org/10.1186/1472-6750-10-61
dc.identifier.urnURN:NBN:no-50991
dc.type.documentTidsskriftartikkel
dc.type.peerreviewedPeer reviewed
dc.identifier.fulltextFulltext https://www.duo.uio.no/bitstream/handle/10852/46811/1/12896_2009_Article_529.pdf
dc.type.versionPublishedVersion
cristin.articleid61


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