Cloning and expression of a soluble μ2M-HLA-A∗0201-heavy chain fusion protein

Abstract

Recombinant soluble HLA molecules may be useful tools for the detection of HLA-specific alloantibodies in the sera of patients awaiting transplantation or peptide-specific T-cell recognition. The production of heterotrimeric HLA in procaryotic expression systems consists of several steps: expression of the heavy chain, expression of β2-microglobulin (β2M), isolation of the recombinant proteins, synthesis of specific peptide-ligands and a final refolding step to assemble the trimeric construct. In order to facilitate these expression and isolation procedures a β2MHLA-A*0201 fusion protein was constructed and expressed in Escherichia coli. Therefore, the extracellular domains (α1, α2 and α3 domains) of HLA-A*0201, were cloned and physically connected to the C-terminus of β2M by a glycine-rich linker. For the detection of the recombinant protein by western blot and direct ELISA a V5 tag was connected to the C-terminus, followed by a histidine-rich sequence for isolation purposes. The proteins were expressed in the inclusion body compartment of E. coli. After isolation by immobilized metal affinity chromatography via the 6xHis-tag, they were finally refolded. An immunological in vitro refold assay involving the monoclonal antibody W6/32 was used to gauge refolding yielding a similar refolding behavior compared to the heterotrimeric protein. Thus, this straight forward strategy has the potential to simplify considerably large scale production of soluble HLA. The chimeric proteins can be used for the induction of epitope-specific CTL responses, as well as for the detection of HLA-specific alloantibodies in the sera of patients.