Protein Expression and Purification   2016 年 123 卷

2016.07.01

Major histocompatibility complex (MHC) class I-related chain A/B (MICA/B) is a type of stress-induced molecule that plays an important role in tumor surveillance. MICA/B shares a similar structure with MHC class I molecules, but MICA/B contains a closed cleft, not an open one, in its N-terminal alpha1 domain. The alpha 1 domain was believed to have no roles in antigen presentation, because the closed cleft provides limited space for binding with known molecules, and the cleft of MICA/B have been reported no known functions. To study the possible function of the cleft located in human MICA/B's alpha 1 domain, we attempted to express the human MICB-?±1 (hMICB-?±1) domain allele protein, which is approximately 20.5 kDa, by utilizing an Escherichia coli (E. coli) secretory pathway. Protein expression was accomplished through the phosphate-limited inducible promoter. After purification using ammonium sulfate precipitation, phenyl hydrophobic Sepharose, SP Sepharose and HisTrap affinity Sepharose, recombinant human MICB-?±1 (rhMICB-?±1) was obtained with 94.3% purity. The binding capacity of rhMICB-?±1 with natural killer group 2, member D (NKG2D) was evaluated in vitro. The results demonstrated that rhMICB-?±1 can be prepared through the E. coli secretory pathway. Purified rhMICB-?±1 protein was able to functionally bind with NKG2D. This method can be further used to obtain functionally active rhMICB-?±1 protein, which can served as the basis for further studies of the possible function of the MICB cleft.