Silkworm expression and sugar profiling of human immune cell surface receptor, KIR2DL1
Silkworm expression and sugar profiling of human immune cell surface receptor, KIR2DL1
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DOI：
10.1016/j.bbrc.2009.07.065
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发表时间：
2009-09-25
影响因子：
3.1
通讯作者：
Maenaka, Katsumi
中科院分区：
文献类型：
--
作者：
Sasaki, Kaori;Kajikawa, Mizuho;Maenaka, Katsumi
免疫细胞表面受体直接参与人类疾病，因此是主要的药物靶点。然而，通常很难获得足够数量的这些受体用于生化和结构研究，因为它们经常需要翻译后修饰，特别是糖修饰。最近，我们建立了杆状病毒BmNPV的杆状病毒表达系统，它直接感染家蚕，是大规模生产重组糖修饰蛋白的有吸引力的宿主。在这里，我们利用BmNPV bacmid表达系统在家蚕中产生了人免疫细胞表面受体，杀伤细胞Ig样受体2DL1(KIR2DL1)。通过直接注射杆状病毒DNA，重组KIR2DL1蛋白得到高效表达，分泌到体液中，并经Ni2+亲和柱层析纯化。对表达条件进行了进一步优化，最终产量为0.2 mg/头。糖谱分析表明，纯化蛋白的N-连接糖组分很少，两种稀释型低聚糖，Manα1-6Man beta 1-4GlcNAc beta 1-4GlcNAc和Man Alpha 1-6Man beta 1-4GlcNAc beta 1-4(Fucα1-6)GlcNAc。这表明该蛋白产物比哺乳动物细胞表达获得的复合糖型产物更为均一。表面等离子体共振分析表明，纯化的KIR2DL1蛋白与人类白细胞抗原CW4配体有特异性结合。此外，圆二色谱显示了合适的二级结构。这些结果清楚地表明，由于相对均一的N-连接糖修饰，家蚕表达系统对于需要翻译后修饰的细胞表面受体的表达以及随后的结构和结合研究是非常有用的。(C)2009 Elsevier Inc.保留所有权利。
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.