Tetramer Production AKTA Pure FPLC

The DNA sequence for each human allele is obtained from the IMGT/HLA Database, codon optimized, a biotin-tag added and cloned into a pET28a plasmid. Each allele or β-2 microglobulin is expressed separately in BL21(DE3) Escherichia coli, purified and subjected to oxidative refolding using a UV-cleavable peptide. The resulting monomer is first purified using ion-exchange chromatography with FPLC, then biotinylated.  The biotinylated monomer is further purified using size-exclusion chromatography with FPLC and stored at -80 °C for subsequent use. Internal controls and verification tests are used at each production step and validation of properly refolded tetramer is completed by staining commercially available characterized PBMCs, where available.