Production and Purification of HLA-E Complexes

Both canonical and Tyr84Cys mutated HLA-E*01:03 heavy chains were expressed in E. coli BL21 (DE3) pLysS competent bacterial cells (Promega, UK) as inclusion bodies and purified according to methods described previously(1 (link), 15 (link), 21 ). Conventional and Tyr84Cys mutated HLA-E*01:03 heavy chains were refolded using standard MHC refolding methods (1 (link), 32 (link)). Following concentration using a Vivaflow 50 (Sartorius, Germany) and Ultra-15 10-kDa cut-off centrifugal units (Sartorius, UK), the samples were subsequently buffered exchanged, using Sephadex G-25 PD10 columns (GE Healthcare, UK) into 10mM Tris for overnight AviTAG biotinylation using the BirA enzyme (Avidity, USA) according to the manufacturer’s instructions. Correctly refolded complexes were purified by size exclusion fast protein liquid chromatography (FPLC) into 20mM Tris pH8 and 100mM NaCl buffer using a HiLoad 16/600 Superdex 75pg column. Correctly folded β2m-HLA-E*01:03-peptide complexes were retrieved, concentrated to 2mg/mL and snap frozen for subsequent tetramer generation.

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Yang H., Rei M., Brackenridge S., Brenna E., Sun H., Abdulhaqq S., Liu M.K., Ma W., Kurupati P., Xu X., Cerundolo V., Jenkins E., Davis S.J., Sacha J.B., Früh K., Picker L.J., Borrow P., Gillespie G.M, & McMichael A.J. (2021). HLA-E restricted Gag specific CD8+ T cells can suppress HIV-1 infection, offering vaccine opportunities. Science immunology, 6(57), eabg1703.

Publication 2021

Vivaflow 50 Sephadex G-25 PD10 columns BirA enzyme

Bacterial Biotinylation Buffer Cells E coli Enzyme Frozen Hla e Inclusion bodies Nacl Peptide Promega Protein Sephadex g 25 Size exclusion chromatography Tetramer Tris

Corresponding Organization : University of Oxford

Other organizations : MRC Human Immunology Unit, Science Oxford, Academy of Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, First Hospital of China Medical University, China Medical University, Oregon National Primate Research Center, Oregon Health & Science University, Chelsea and Westminster Hospital, Imperial College London

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Variable analysis

independent variables

Expression of canonical and Tyr84Cys mutated HLA-E*01:03 heavy chains

dependent variables

Refolding of conventional and Tyr84Cys mutated HLA-E*01:03 heavy chains
Purification of correctly refolded β2m-HLA-E*01:03-peptide complexes

control variables

Expression of HLA-E*01:03 heavy chains in E. coli BL21 (DE3) pLysS competent bacterial cells
Purification of HLA-E*01:03 heavy chains as inclusion bodies
Standard MHC refolding methods
Concentration and buffer exchange using Vivaflow 50, Ultra-15 10-kDa cut-off centrifugal units, and Sephadex G-25 PD10 columns
Overnight AviTAG biotinylation using the BirA enzyme
Purification of correctly refolded complexes by size exclusion fast protein liquid chromatography (FPLC) into 20mM Tris pH8 and 100mM NaCl buffer using a HiLoad 16/600 Superdex 75pg column

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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