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J Immunol Methods. 2005 Nov 30;306(1-2):51-67. Epub 2005 Sep 12.

High-level bacterial secretion of single-chain alphabeta T-cell receptors.

Author information

1
Department of Microbiology and Immunology and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94304, USA. jmaynard@umn.edu

Abstract

While numerous antibody-antigen systems have been structurally characterized, studies of structurally analogous T-cell receptor MHC systems have lagged behind largely due to the lack of a general TCR expression system. Efforts to develop bacterial systems have resulted in low yields (< 0.5 mg/l) of active material which is prone to proteolysis and aggregation. Here we report a strategy to secrete folded, soluble single chain T-cell receptors (scTCR) in the Escherichia coli periplasm using three representative alphabeta TCRs (172.10, 1934.4/c19 and 2B4). Shake flask yields between 0.5 and 30 mg/l active, purified material were attained for all TCRs studied and found to depend on the introduction of solubility-increasing amino acid substitutions, skp chaperone co-expression and C-terminal fusion to a human kappa constant domain in the context of a tightly regulated expression vector. This system will greatly enable crystallographic, thermodynamic and other biophysical analyses of TCRs which require large quantities of homogeneous material.

PMID:
16198365
DOI:
10.1016/j.jim.2005.07.022
[Indexed for MEDLINE]

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