title: High-level bacterial secretion of single-chain alphabeta T-cell receptors.
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.
authors.affiliation: Department of Microbiology and Immunology and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94304, USA. jmaynard@umn.edu
authors.forename: Jennifer
authors.initials: J
authors.lastname: Maynard
authors.forename: Erin J
authors.initials: EJ
authors.lastname: Adams
authors.forename: Michelle
authors.initials: M
authors.lastname: Krogsgaard
authors.forename: Karin
authors.initials: K
authors.lastname: Petersson
authors.forename: Corey W
authors.initials: CW
authors.lastname: Liu
authors.forename: K Christopher
authors.initials: KC
authors.lastname: Garcia
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D000596:Amino Acids; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins