TCR chains were expressed individually in E. coli Rosetta cells (Novagen/Merck, Germany) as insoluble inclusion bodies and purified largely as described.13 (link) Briefly, TCRs were produced by refolding in 8M urea and slow dialysis. A molar ratio of 1.2:1 alpha to beta chain was mixed under denaturing conditions and dialyzed for up to 48 hours in 10 mM Tris pH 8.0 before performing ion exchange chromatography (HiTrap MonoQ; Cytiva, USA), followed by size-exclusion chromatography (HR 16/600 or HR 10/300 columns; GE Healthcare, UK). pHLA was produced similarly but with a 1:2 molar ratio of HLA alpha chain to β-2-microglobulin and an excess added of the MAGE-A10-GLY 9-mer peptide (>98% purity; Peptide Protein Research, UK) with up to 60 hours of refolding with dialysis, followed by ion exchange chromatography and size-exclusion chromatography. TCR–pHLA complexes were formed by mixing equimolar quantities of pHLA and TCR, and final purification by size-exclusion chromatography in phosphate-buffered saline.
Protocol detail
TCR and pHLA Expression and Purification
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Cells
Dialysis
E coli
Inclusion bodies
Ion exchange chromatography
Mage a10
Molar
Peptide
Phosphate
Protein
Saline
Size exclusion chromatography
Tris
Urea
β 2 microglobulin
Corresponding Organization : Adaptimmune (United Kingdom)
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Variable analysis
independent variables
- Expression of TCR chains individually in E. coli Rosetta cells
- Refolding of TCRs in 8M urea and slow dialysis
- Mixing of TCR alpha and beta chains in a molar ratio of 1.2:1 under denaturing conditions and dialysis for up to 48 hours
- Production of pHLA with a 1:2 molar ratio of HLA alpha chain to β-2-microglobulin and excess MAGE-A10-GLY 9-mer peptide, followed by refolding with dialysis for up to 60 hours
- Mixing of equimolar quantities of pHLA and TCR to form TCR–pHLA complexes
- Purification of TCRs and pHLA complexes using ion exchange chromatography and size-exclusion chromatography
- Tris pH 8.0 buffer used for dialysis of TCR-alpha and -beta chains
- Phosphate-buffered saline used for final purification of TCR–pHLA complexes
- No explicit mention of positive or negative controls
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