Major Histocompatibility Complex class I-related chain molecules A
(MICA) and receptor Natural killer group 2 member D (NKG2D) are important membrane
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 dialysis buffer was selected based on the theoretical isoelectric points of
MICA and NKG2D which were 5.0 and 5.2 respectively. 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 pancreatic 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.
Keywords: Chaotropic reagent, Inclusion body, MICA, NKG2D, refolding, solubilization.
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