Protein Expression and Purification, Vol.20, No.1, 10-20, 2000
Expression in Escherichia coli, folding in vitro, and characterization of the carbohydrate recognition domain of the natural killer cell receptor NKR-P1A
NKR-P1A is a homodimeric type II transmembrane protein of the C-type lectin family found on natural killer (NK) cells and NH-like T cells and is an activator of cytotoxicity, Toward structure determination by NMR, the recombinant carbohydrate-recognition domain (CRD) of NKR-P1A has been expressed in high-yield in Escherichia coil and folded in vitro, The purified protein behaves as a monomer in size-exclusion chromatography and is bound by the conformation-sensitive antibody, 3.2.3, indicating a folded structure. A polypeptide tag at the N-terminus is selectively cleaved from the CRD after limited trypsin digestion in further support of a compact folded structure. The disulfide bonds have been identified by peptide mapping and electrospray mass spectrometry, These are characteristic of a long form CRD, The 1D NMR spectrum of the unlabeled CRD and the 2D HSQC spectrum of the N-15-labeled CRD are those of a folded protein. Chemical shifts of H-alpha and NH protons indicate a considerable amount of beta-strand structure. Successful folding in the absence of Ca2+, coupled with the lack of chemical shift changes upon addition of Ca2+, suggests that the NKR-P1A-CRD may not be a Ca2+-binding protein.