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

Maynard, Jennifer; Adams, Erin J.; Krogsgaard, Michelle; Lindkvist, Karin LU ; Liu, Corey W. and Garcia, K. Christopher (2005) In Journal of Immunological Methods 306(1-2). p.51-67
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... (More)

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.

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author
publishing date
type
Contribution to journal
publication status
published
keywords
Crystallography, Recombinant expression, scTCR, skp, TCR
in
Journal of Immunological Methods
volume
306
issue
1-2
pages
17 pages
publisher
Elsevier
external identifiers
  • scopus:28444442005
ISSN
0022-1759
DOI
10.1016/j.jim.2005.07.022
language
English
LU publication?
no
id
85316f9f-7041-4937-a58e-6fe28964594e
date added to LUP
2017-02-15 15:29:05
date last changed
2017-07-30 05:21:39
@article{85316f9f-7041-4937-a58e-6fe28964594e,
  abstract     = {<p>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 (&lt; 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.</p>},
  author       = {Maynard, Jennifer and Adams, Erin J. and Krogsgaard, Michelle and Lindkvist, Karin and Liu, Corey W. and Garcia, K. Christopher},
  issn         = {0022-1759},
  keyword      = {Crystallography,Recombinant expression,scTCR,skp,TCR},
  language     = {eng},
  month        = {11},
  number       = {1-2},
  pages        = {51--67},
  publisher    = {Elsevier},
  series       = {Journal of Immunological Methods},
  title        = {High-level bacterial secretion of single-chain αβ T-cell receptors},
  url          = {http://dx.doi.org/10.1016/j.jim.2005.07.022},
  volume       = {306},
  year         = {2005},
}