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Title: Expression in Escherichia coli, folding in vitro, and characterization of the carbohydrate recognition domain of the natural killer cell receptor NKR-P1A.
Authors: Kogelberg, H
Lawson, AM
Muskett, FW
Carruthers, RA
Feizi, T
First Published: Oct-2000
Citation: PROTEIN EXPR PURIF, 2000, 20 (1), pp. 10-20
Abstract: 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.
DOI Link: 10.1006/prep.2000.1257
ISSN: 1046-5928
Type: Journal Article
Appears in Collections:Published Articles, Dept. of Biochemistry

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