화학공학소재연구정보센터
Protein Expression and Purification, Vol.123, 83-89, 2016
Expression and purification of human MHC class I-related chain molecule B-alpha 1 domain
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 alphal 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-alpha 1 (hMICB-alpha 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-alpha 1 (rhMICB-alpha 1) was obtained with 94.3% purity. The binding capacity of rhMICB-alpha 1 with natural killer group 2, member D (NKG2D) was evaluated in vitro. The results demonstrated that rhMICB-alpha 1 can be prepared through the E. coli secretory pathway. Purified rhMICB-alpha 1 protein was able to functionally bind with NKG2D. This method can be further used to obtain functionally active rhMICB-alpha 1 protein, which can served as the basis for further studies of the possible function of the MICB cleft. (C) 2016 Elsevier Inc. All rights reserved.