期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 387, 期 3, 页码 575-580出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2009.07.065
关键词
Bacmid; BmNPV; Bombyx mori; Silkworm; Baculovirus expression system; Killer cell Ig-like receptor; N-glycosylation
资金
- Ministry of Education, Culture, Sports, Science and Technology [21370050]
- Ministry of Health
- Japan Society for the Promotion of Science for young scientists
- Grants-in-Aid for Scientific Research [21370050] Funding Source: KAKEN
Immune cell surface receptors are directly involved in human diseases, and thus represent major drug targets. However, it is generally difficult to Obtain Sufficient amounts of these receptors for biochemical and structural Studies because they often require post translational modifications, especially sugar modification Recently, we have established a bacmid expression system for the baculovirus BmNPV, which directly infects silkworms, an attractive host for the large-scale production of recombinant sugar-modified proteins. Here we produced the human immune cell surface receptor, killer cell Ig-like receptor 2DL1 (KIR2DL1), by Using the BmNPV bacmid expression system, in silkworms. By the direct injection of the bacmid DNA, the recombinant KIR2DL1 protein was efficiently expressed, secreted into body fluids, and purified by Ni2+ affinity column chromatography. We further optimized the expression conditions, and the final yield was 0.2 mg/larva. The sugar profiling revealed that the N-linked Sugars of the purified protein comprised very few components, two paucimannose-type oligosaccharides, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc and Man alpha 1-6Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc. This revealed that the protein product was Much more homogeneous than the complex-sugar type Product obtained by mammalian cell expression. The Surface plasmon resonance analysis demonstrated that the purified KIR2DL1 protein exhibited specific binding to the HLA-Cw4 ligand. Moreover, the CD spectrum showed the proper secondary Structure. These results clearly Suggested that the silkworm expression system is quite useful for the expression of cell surface receptors that require posttranslational modifications, as well as for then-structural and binding studies, due to the relatively homogeneous N-linked sugar modifications. (C) 2009 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据