Expression and purification of the angiogenesis inhibitor 16-kDa prolactin fragment from insect cells
Expression and purification of the angiogenesis inhibitor 16-kDa prolactin fragment from insect cells
复制标题
DOI：
10.1016/s1046-5928(02)00639-3
复制
发表时间：
2003-04-01
影响因子：
1.6
通讯作者：
Lin, SH
中科院分区：
文献类型：
--
作者：
Galfione, M;Luo, WP;Lin, SH
催乳素 (16-kDa PRL) 的 16-kDa 片段源自 23-kDa PRL 的蛋白水解裂解，具有抗血管生成活性。先前的研究表明，由细菌产生的重组16-kDa PRL通常含有内毒素，对内皮细胞具有细胞毒性，并且由于其从包涵体重折叠的变化而导致其生物活性变化。这些问题限制了重组 16-kDa PRL 的使用。为了提高重组 16-kDa PRL 的产生，我们使用杆状病毒表达系统在 Sf9 昆虫细胞中表达 16-kDa PRL。将人 PRL 基因的信号序列和七个组氨酸的密码子分别添加到 16-kDa PRL cDNA 构建体的 N 端和 C 端。在细胞沉淀和培养基中均检测到重组 16-kDa PRL。使用镍亲和层析从 4 x 107 个感染细胞的细胞沉淀中分离出约 0.28 mg 纯化的蛋白质。 16 千道尔顿 PRL 经过翻译后修饰，在 SDS PAGE 上表观分子量为 16 和 18 kDa。用肽基-N-糖苷酶消化后，18-kDa 蛋白的水平显着降低，表明异质性是由于 16-kDa PRL 的糖基化造成的。 N端序列分析证实这两种蛋白都是人16-kDa PRL，并且信号序列在与人PRL相同的位置被切割。与其作为血管生成抑制剂的作用一致，纯化的重组 16-kDa PRL 抑制内皮细胞的增殖，其效力类似于之前报道的大肠杆菌中产生的蛋白质。这种在 Sf9 细胞中表达的 16-kDa PRL 是一种有用的试剂，可用于功能研究以及其受体的纯化和鉴定。 (C) 2003 年爱思唯尔科学（美国）。版权所有。
The 16-kDa fragment of prolactin (16-kDa PRL), derived from proteolytic cleavage of 23-kDa PRL, was shown to have antiangiogenic activity. Previous studies have shown that recombinant 16-kDa PRL produced from bacteria often contained endotoxins, which are cytotoxic to endothelial cells, and varied in its biological activity due to changes in its refolding from inclusion bodies. These problems limited the use of recombinant 16-kDa PRL. To improve the generation of recombinant 16-kDa PRL, we expressed 16-kDa PRL in Sf9 insect cells using a baculoviral expression system. The signal sequence of the human PRL gene and codons for seven histidines were added to the N- and C-termini, respectively, of the 16-kDa PRL cDNA construct. Recombinant 16-kDa PRL was detected in both the cell pellet and the medium. About 0.28 mg purified protein was isolated from the cell pellet of 4 x 107 infected cells using nickel affinity chromatography. Sixteen kilodalton PRL was posttranslationally modified with apparent molecular weights of 16 and 18 kDa on SDS PAGE. The level of 18-kDa protein was significantly reduced after digestion with peptidyl-N-glycosidase, suggesting that the heterogeneity was due to glycosylation of 16-kDa PRL. N-terminal sequence analysis confirmed the fact that both proteins were human 16-kDa PRL and the signal sequences were cleaved at the same position as that of human PRL. Consistent with its role as an angiogenesis inhibitor, purified recombinant 16-kDa PRL inhibits the proliferation of endothelial cells with a potency similar to that previously reported for the protein generated in Escherichia coli. This 16-kDa PRL expressed in Sf9 cells is a useful reagent for functional studies and for the purification and identification of its receptor. (C) 2003 Elsevier Science (USA). All rights reserved.