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 NK-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 coli 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 (15)N-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 Ca(2+), coupled with the lack of chemical shift changes upon addition of Ca(2+), suggests that the NKR-P1A-CRD may not be a Ca(2+)-binding protein.
Kogelberg
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H
The Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Campus, Harrow, Middlesex, United Kingdom. h.kogelberg@ic.ac.uk
Lawson
A M
AM
Muskett
F W
FW
Carruthers
R A
RA
Feizi
T
T
D000595:Amino Acid Sequence; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D007694:Killer Cells, Natural / Q000378:metabolism*; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D055651:NK Cell Lectin-Like Receptor Subfamily B; D019906:Nuclear Magnetic Resonance, Biomolecular; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000954:Antigens, Surface; D017931:DNA Primers; D037181:Lectins, C-Type; D055651:NK Cell Lectin-Like Receptor Subfamily B; D011971:Receptors, Immunologic; D011994:Recombinant Proteins