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

J Immunol Methods. 2005 Nov 30;306(1-2):51-67. doi: 10.1016/j.jim.2005.07.022. Epub 2005 Sep 12.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Amino Acids / chemistry
  • Crystallization
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Protein Conformation
  • Protein Engineering
  • Protein Folding
  • Receptors, Antigen, T-Cell, alpha-beta / biosynthesis*
  • Receptors, Antigen, T-Cell, alpha-beta / chemistry
  • Receptors, Antigen, T-Cell, alpha-beta / genetics
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification

Substances

  • Amino Acids
  • Receptors, Antigen, T-Cell, alpha-beta
  • Recombinant Proteins