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Efficient In Vitro Refolding and Characterization of Major Histocompati- bility Complex Class I-Related Chain Molecules A (MICA) and Natural Killer Group 2 Member D (NKG2D) Expressed in E. coli

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dc.contributor.author Zhao, Xin
dc.contributor.author Acheampong, Desmond Omane
dc.contributor.author Wang, Youfu
dc.contributor.author Tang, Mingying
dc.contributor.author Xie, Wei
dc.contributor.author Chen, Zhiguo
dc.contributor.author Wang, Min
dc.contributor.author Zhang, Juan
dc.date.accessioned 2023-09-29T15:16:46Z
dc.date.available 2023-09-29T15:16:46Z
dc.date.issued 2015
dc.identifier.uri http://hdl.handle.net/123456789/8725
dc.description.abstract Major Histocompatibility Complex class I-related chain molecules A (MICA) and receptor Natural killer group 2 member D (NKG2D) are important mem- brane proteins with immunosurveillance properties which could serve as therapeutic targets for immunotherapy. However, expression of MICA and NKG2D in E coli often leads to the formation of inclusion bodies. Here, we present simple, inexpensive and convenient protocol for the solubilization and refolding of inclusion bodies of MICA and NKG2D expressed in E coli. The inclusion bodies were firstly dissolved in strong chaotropic reagent (8M urea) and subsequently purified by immobilized-metal affinity column. The denatured MICA/NKG2D was refolded by gradually removing both denaturant (8M urea) and imidazole via dialysis in dialysis buffer of pH 7.4. The appropriate pH of the di- alysis buffer was selected based on the theoretical isoelectric points of MICA and NKG2D which were 5.0 and 5.2 respec- tively. The folded MICA and NKG2D demonstrated the capacity to bind to recombinant NKG2D and MICA respectively by ELISA, Western blot and Surface Plasmon Resonance (SPR) assays. Additionally, the folded MICA and NKG2D demonstrated significant binding to NKG2D-positive Human leukemic cell line U937 and MICA-positive Human pancre- atic carcinoma, epithelial-like cell line (PANC-1) respectively, suggesting successful refolding. Successful refolding was further confirmed by Circular Dichroism spectroscopy (CD). We have successfully dissolved, refolded and characterized inclusion bodies of MICA/NKG2D expressed in E. coli using simple, inexpensive and convenient protocol which can be carried out in laboratories under-resourced. en_US
dc.language.iso en en_US
dc.publisher University of Cape Coast en_US
dc.subject Inclusion body en_US
dc.subject MICA en_US
dc.subject NKG2D en_US
dc.subject refolding en_US
dc.subject solubilization en_US
dc.subject chaotropic reagent en_US
dc.title Efficient In Vitro Refolding and Characterization of Major Histocompati- bility Complex Class I-Related Chain Molecules A (MICA) and Natural Killer Group 2 Member D (NKG2D) Expressed in E. coli en_US
dc.type Article en_US


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