Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display

Gunnarsen, Kristin Støen; Kristinsson, Solveig Gunn; Justesen, Sune; Frigstad, Terje; Buus, Søren; Bogen, Bjarne; Sandlie, Inger; Løset, Geir Åge

doi:10.1038/srep01162

Cited by 25 publications

(35 citation statements)

References 54 publications

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“…To elucidate the intrinsic pMHC binding properties of the TCRs 364 and 380, we first expressed and purified both TCRs in a single-chain TCR (scTCR) format containing stabilizing mutations (P50 TRAV , L50 TRBV , F103 TRBV , and P2 linker ) (13,14). We then performed surface plasmon resonance (SPR) studies employing recombinant, soluble TCRs and pMHCs.…”

Section: Generation Of α-Gliadin-specific T Cell Clones and Tcr Clonimentioning

confidence: 99%

A TCRα framework–centered codon shapes a biased T cell repertoire through direct MHC and CDR3β interactions

Gunnarsen¹,

Høydahl²,

Risnes³

et al. 2017

JCI Insight

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Section: Generation Of α-Gliadin-specific T Cell Clones and Tcr Clonimentioning

confidence: 99%

A TCRα framework–centered codon shapes a biased T cell repertoire through direct MHC and CDR3β interactions

Gunnarsen¹,

Høydahl²,

Risnes³

et al. 2017

JCI Insight

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“…In contrast, the pIII-derived clone, 57AE06 (using IGHV3) was expressed extremely well, but bound poorly to the antigen, indicating selection largely based on favorable expression characteristics. Although expression and stability are closely connected4647, it did not appear to be the case with this clone. This was even more pronounced for the other pIII clone, which had a high thermal stability, but was expressed at very low level.…”

Section: Discussionmentioning

confidence: 71%

Multivalent pIX phage display selects for distinct and improved antibody properties

Høydahl

Nilssen

Gunnarsen

et al. 2016

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Phage display screening readily allows for the identification of a multitude of antibody specificities, but to identify optimal lead candidates remains a challenge. Here, we direct the antibody-capsid fusion away from the signal sequence-dependent secretory SEC pathway in E. coli by utilizing the intrinsic signal sequence-independent property of pIX to obtain virion integration. This approach was combined with the use of an engineered helper phage known to improve antibody pIX display and retrieval. By direct comparison with pIII display, we demonstrate that antibody display using this pIX system translates into substantially improved retrieval of desired specificities with favorable biophysical properties in de novo selection. We show that the effect was due to less E. coli host toxicity during phage propagation conferred by the lack of a signal sequence. This pIX combinatorial display platform provides a generic alternative route for obtaining good binders with high stability and may thus find broad applicability.

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“…We thought it likely that some periplasmic factor must be assisting LacZ folding. Because previous studies have implicated the periplasmic chaperones skp and fkpA in the folding of heterologous proteins (23)(24)(25)(26)(27)(28)(29), we chose to see what effect disruption of these genes might have on the ␤-galactosidase activity of a strain expressing the malF-lacZ102 fusion in the absence of DsbA. While disruption of skp did not decrease ␤-galactosidase activity, we found that disruption of fkpA reduced it by a factor of 2.6 (Fig.…”

Section: Resultsmentioning

confidence: 97%

Folding LacZ in the Periplasm of Escherichia coli

et al. 2014

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b Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematically characterize the nonjamming fusions and determine their ultimate localization. We report that LacZ can be fully translocated into the periplasm, where it is toxic. We show that this toxicity is likely due to LacZ misfolding and that, in the absence of the periplasmic disulfide bond catalyst DsbA, LacZ folds in the periplasm. Using the novel phenotype of periplasmic ␤-galactosidase activity, we show that the periplasmic chaperone FkpA contributes to LacZ folding in this nonnative compartment. We propose that targeted, nonjamming LacZ fusions may be used to further study folding and targeting in the periplasm of Escherichia coli.

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Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display

Cited by 25 publications

References 54 publications

A TCRα framework–centered codon shapes a biased T cell repertoire through direct MHC and CDR3β interactions

A TCRα framework–centered codon shapes a biased T cell repertoire through direct MHC and CDR3β interactions

Multivalent pIX phage display selects for distinct and improved antibody properties

Folding LacZ in the Periplasm of Escherichia coli

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