High-level bacterial secretion of single-chain αβ T-cell receptors
(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
- Maynard, Jennifer ; Adams, Erin J. ; Krogsgaard, Michelle ; Lindkvist, Karin LU ; Liu, Corey W. and Garcia, K. Christopher
- publishing date
- 2005-11-30
- 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
-
- pmid:16198365
- 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
- 2024-10-14 00:02:04
@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 (< 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}}, keywords = {{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}}, doi = {{10.1016/j.jim.2005.07.022}}, volume = {{306}}, year = {{2005}}, }