Lab Item Demo 1

Tetramer Production

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.

Data
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.