A Combination Strategy for Construction of Peptide-β2m-H-2Kb Single Chain with Overlap Extension PCR and One-Step Cloning.

The time-consuming and high-cost preparation of soluble peptide-major histocompatibility complexes (pMHC) currently limits their wide uses in monitoring antigen-specific T cells. The single-chain trimer (SCT) of peptide-β2m-MHC class I heavy chain was developed as an alternative strategy, but its gene fusion is hindered in many cases owing to the incompatibility between the multiple restriction enzymes and the restriction endonuclease sites of plasmid vectors. In this study, overlap extension PCR and one-step cloning were adopted to overcome this restriction. The SCT gene of the OVA₂₅₇₋₂₆₄ peptide-(GS₄)₃-β2m-(GS₄)₄-H-2Kb heavy chain was constructed and inserted into plasmid pET28a by overlap extension PCR and one-step cloning, without the requirement of restriction enzymes. The SCT protein was expressed in Escherichia coli, and then purified and refolded. The resulting H-2Kb/OVA₂₅₇₋₂₆₄ complex showed the correct structural conformation and capability to bind with OVA₂₅₇₋₂₆₄-specific T-cell receptor. The overlap extension PCR and one-step cloning ensure the construction of single-chain MHC class I molecules associated with random epitopes, and will facilitate the preparation of soluble pMHC multimers.