High-level production of a secreted, heterodimeric αβ murine T-cell receptor in Escherichia coli

Golden, Alex; Khandekar, Sanjay S.; Osburne, Marcia S.; Kawasaki, Ernest S.; Reinherz, Ellis L.; Grossman, Trudy H.

doi:10.1016/s0022-1759(97)00106-3

Cited by 14 publications

(4 citation statements)

References 16 publications

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“…Both prokaryotic and eukaryotic expression systems have proven to be valid sources of soluble TCRs. For prokaryotic systems, refolding has an efficient alternative in periplasmic expression, where the oxidative environment allows the formation of disulfide bonds . Periplasmic TCR expression is enhanced if the bacterial secretory pathway is supported by overexpression of folding chaperone proteins , and higher stability of the expressed TCR correlates with the fraction of the active product .…”

Section: Discussionmentioning

confidence: 99%

Stabilization of soluble high‐affinity T‐cell receptor with de novo disulfide bonds

et al. 2019

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Soluble T‐cell receptors (TCRs) have recently gained visibility as target‐recognition units of anticancer immunotherapeutic agents. Here, we improved the thermal stability of the well‐expressed high‐affinity A6 TCR by introducing pairs of cysteines in the invariable parts of the α‐ and β‐chain. A mutant with a novel intradomain disulfide bond in each chain also tested superior to the wild‐type in the accelerated stability assay. Binding of the mutant to the soluble cognate peptide (cp)–MHC and to the peptide‐loaded T2 cell line was equal to the wild‐type A6 TCR. The same stabilization motif worked efficiently in TCRs with different specificities, such as DMF5 and 1G4. Altogether, the biophysical properties of the soluble TCR molecule could be improved, without affecting its expression level and antigen‐binding specificity.

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Section: Discussionmentioning

confidence: 99%

Stabilization of soluble high‐affinity T‐cell receptor with de novo disulfide bonds

et al. 2019

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“…46 -51 In the oxidizing milieu of the periplasm of E. coli, folding and formation of disulfide bonds may also be achieved, but again with comparably low yields. 52 In contrast, expression in the cytosol of bacteria may yield much larger amounts of protein, but the TCR chains have to be refolded in vitro from insoluble inclusion bodies. [53][54][55][56][57] Considerably different constructs have been used.…”

Section: Soluble Tcr Moleculesmentioning

confidence: 99%

T-Cell-Mediated Autoimmunity

Dornmair

Goebels²,

Weltzien

et al. 2003

The American Journal of Pathology

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Histological samples of autopsy or biopsy tissue provide the best available evidence that autoreactive T cells are involved in the immunopathogenesis of many autoimmune diseases. However, morphology alone does not provide information on the antigen-specific T-cell receptor (TCR) of these cells, let alone on their antigen specificity. In this review article we discuss a number of emerging possibilities for identifying TCR sequences directly from biopsy tissue. We also review the methods for expressing presumably autoreactive TCR molecules and speculate on how the expressed TCR might be used to identify target antigens. Such information should eventually provide new insights into disease pathogenesis which lead to better therapies.

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“…Periplasmic expression of TCR, as an alternative screening method, has yet to prove effective [5][6][7]. Other strategies to produce soluble proteins have included expression within the bacterial cytoplasm.…”

Section: Introductionmentioning

confidence: 99%

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of Escherichia coli trxB gor Mutants

et al. 2010

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Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed alpha and beta chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.

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High-level production of a secreted, heterodimeric αβ murine T-cell receptor in Escherichia coli

Cited by 14 publications

References 16 publications

Stabilization of soluble high‐affinity T‐cell receptor with de novo disulfide bonds

Stabilization of soluble high‐affinity T‐cell receptor with de novo disulfide bonds

T-Cell-Mediated Autoimmunity

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of Escherichia coli trxB gor Mutants

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