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重组人P--选凝素及P--选凝素糖蛋白配基--1的表达与鉴定

裴新辉, 林志新, 耿建国

上海交通大学生命科学与技术学院.上海 200240；中科院上海生命科学研究院生物化学与细胞生物学研究所分子细胞生物学实验室.上海 200032

摘要

P--选凝素表达于血管内皮细胞及血小板膜上,它可以与白细胞膜表面的P--选凝素糖蛋白配基--1(P--selectin glycoprotein ligand--1,PSGL--1)相互作用,在炎症过程中介导白细胞的滚动并启动随后的白细胞迁移级联过程。我们构建了重组人野生型可溶性P--选凝素及其钙离子结合位点突变体,同时构建了重组PSGL--1免疫球蛋白融合分子(PSGL--1--Rg),并应用昆虫杆状病毒表达系统在Sf9细胞中表达这些重组蛋白,最后用镍金属螯和柱或Protein A亲和柱予以纯化。结果显示,用该系统表达的P--选凝素或PSGL--1是有活性的,但是P--选凝素的4个钙离子结合位点突变体却没有活性。该研究证明了P--选凝素钙离子结合位点在其与配基相互作用中的重要性。

关键词： P-选凝素; P-选凝素糖蛋白配基-1; 昆虫杆状病毒; 昆虫细胞Sf9

Expression and identification of recombinant P--selectin and P--selectin glycoprotein ligand--1

Pei Xinhui, Lin Zhixin, Geng Jianguo

College of Life Science and Biotechnology,Shanghai Jiaotong University.Shanghai 200240；China

Abstract

P-selectin, one of the membrane proteins, expresses on platelet and endothelia and interacts with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocyte membrane. This interaction mediates leukocytes rolling on endothelial membrane and then induces leukocyte recruitment to the site of infection or tissue injury. In the present study, we constructed the recombinant wild type human P-selectin, its calcium-binding sites mutants and recombinant PSGL-1-globulin (PSGL-1-Rg). They expressed in Sf9 cells by using the baculovirus expression system and were purified by Talon~(TM) metal or Protein A affinity chromatography. The results showed that the recombinant PSGL-1-Rg interacted with recombinant wild type P-selectin and two P-selectin mutants with 2 calcium-binding sites mutation respectively, but could not bind to the P-selectin mutant with all 4 calcium-binding sites mutation. Therefore, we verified the importance of P-selectin calcium-binding sites for its interaction with PSGL-1.

Key words: P-selectin;P-selectin glycoprotein ligand-1;baculovirus;Sf9 cell

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引用本文：

裴新辉, 林志新, 耿建国. 重组人P--选凝素及P--选凝素糖蛋白配基--1的表达与鉴定[J]. 生理学报 2008; 60 (4): 520-524.

Pei Xinhui, Lin Zhixin, Geng Jianguo. Expression and identification of recombinant P--selectin and P--selectin glycoprotein ligand--1. Acta Physiol Sin 2008; 60 (4): 520-524 (in Chinese with English abstract).