High-level bacterial secretion of single-chain αβ T-cell receptors

Research output: Contribution to journalArticle

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 αβ 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.

Details

Authors
  • Jennifer Maynard
  • Erin J. Adams
  • Michelle Krogsgaard
  • Karin Lindkvist
  • Corey W. Liu
  • K. Christopher Garcia
External organisations
  • Stanford University
Research areas and keywords

Keywords

  • Crystallography, Recombinant expression, scTCR, skp, TCR
Original languageEnglish
Pages (from-to)51-67
Number of pages17
JournalJournal of Immunological Methods
Volume306
Issue number1-2
Publication statusPublished - 2005 Nov 30
Publication categoryResearch
Peer-reviewedYes
Externally publishedYes