Kogelberg H, Lawson A M, Muskett F W, Carruthers R A, Feizi T
The Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Campus, Harrow, Middlesex, United Kingdom.
Protein Expr Purif. 2000 Oct;20(1):10-20. doi: 10.1006/prep.2000.1257.
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.
NKR-P1A是一种C型凝集素家族的同二聚体II型跨膜蛋白，存在于自然杀伤（NK）细胞和NK样T细胞上，是细胞毒性的激活剂。为了通过核磁共振确定其结构，NKR-P1A的重组碳水化合物识别结构域（CRD）已在大肠杆菌中高表达并在体外折叠。纯化后的蛋白在尺寸排阻色谱中表现为单体，并与构象敏感抗体3.2.3结合，表明其具有折叠结构。在有限的胰蛋白酶消化后，N端的多肽标签从CRD中被选择性切割，进一步支持了其紧密折叠结构。通过肽图谱和电喷雾质谱确定了二硫键。这些是长形式CRD的特征。未标记CRD的一维核磁共振谱和（15）N标记CRD的二维HSQC谱均显示为折叠蛋白的谱图。Hα和NH质子的化学位移表明存在大量的β链结构。在没有Ca2+的情况下成功折叠，以及添加Ca2+后化学位移没有变化，表明NKR-P1A-CRD可能不是一种Ca2+结合蛋白。
