Production of a soluble disulfide bond-linked TCR in the cytoplasm of Escherichia coli trxB gor mutants.
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.
Immunocore Ltd, 57c Milton Park, Abingdon, Oxfordshire OX14 4RX, UK.
Nathaniel
N
Liddy
Peter E
PE
Molloy
Alan D
AD
Bennett
Jonathan M
JM
Boulter
Bent K
BK
Jakobsen
Yi
Y
Li
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005980:Glutathione Reductase / Q000235:genetics* / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D020349:Surface Plasmon Resonance; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D011948:Receptors, Antigen, T-Cell; C072721:Skp protein, E coli; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase; D019704:Protein Disulfide-Isomerases; C526649:dsbC protein, E coli