Expression and purification of the minor histocompatibility antigen, HA-1H generated in Escherichia coli.
The minor histocompatibility antigen HA-1H is a potential immunotherapeutic molecule. It can be used as a target for graft versus leukaemia reactions to eliminate residual HA-1H expressing leukaemic cells in patients following haemopoietic stem cell transplantation, whereby HA-1H primed donor cells can be transferred into a patient via adoptive immunotherapy. However, thus far only synthetic peptides corresponding to a HLA-A *0201 restricted HA-1H epitope have been used to generate HA-1H specific T cells. We are the first laboratory to clone, express and purify a region of HA-1H using an Escherichia coli expression system. The recombinant HA-1H protein was purified under denaturing conditions and the affinity purification tag removed using thrombin to remove non-specific amino acids. The 92 amino acid recombinant protein was characterised by mass spectrometry. Our rationale is that by using a recombinant HA-1H protein rather than peptide, HA-1H specific T cells may be generated from presentation of multiple HA-1H epitopes complexed in different HLA molecules. A multi-epitope approach may have wider applicability and maybe more effective at leukaemia control. The recombinant HA-1H protein may also be used as a research tool to identify novel CD4(+) helper T cell and CD8(+) cytotoxic T cell epitopes.
Etto
Tamara L
TL
Immunotherapy Research Group, Bone Marrow Transplant Unit, Alfred Hospital, Melbourne, Australia. t.etto@alfred.org.au
Stewart
Lisbeth A
LA
Nguyen
Thi H O
TH
Williamson
Nicholas A
NA
Purcell
Anthony W
AW
Schwarer
Anthony P
AP
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015778:Minor Histocompatibility Antigens / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein
C110615:HA-1 antigen; D015778:Minor Histocompatibility Antigens; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine