High-level bacterial expression, purification and characterization of human calreticulin.
High-level bacterial expression, purification and characterization of human calreticulin.
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人钙网蛋白的高水平细菌表达、纯化和表征。
DOI：
10.1093/protein/4.8.981
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Hoch,SO
中科院分区：
文献类型：
--
作者：
Rokeach,LA;Haselby,JA;Hoch,SO
为了研究其细胞功能及其在自身免疫性疾病发病机制中的作用，我们通过细菌表达了人钙网蛋白，一种内质网中主要的钙结合蛋白和人自身抗原。这是首次报道钙网蛋白在不同来源的异种表达。重组钙网蛋白占可溶性大肠杆菌蛋白的约32%，通过离子交换和疏水液相色谱纯化得到明显的均匀性。与真诚蛋白一样，重组钙网蛋白与钙结合，并在与Zn2+结合时改变其构象。我们认为这是一个强有力的迹象表明，折叠的。大肠杆菌表达的钙网蛋白与真正的蛋白非常相似，如果不是完全相同的话。此外，细菌表达的钙网蛋白容易与抗人抗体和抗兔抗体发生反应，抗重组钙网蛋白抗体与HeLa钙网蛋白发生免疫反应。该表达系统的可用性将使我们能够在钙网蛋白的结构-功能研究中进行位点特异性和缺失突变分析。
To investigate its cellular function and role in autoimmune disease pathogenesis, we have bacterlally expressed human calreticulin, a major calcium-binding protein in the endoplasmic reticulum and a human autoantigen. This is the First report describing the heterologous expression of calreticulin from any source. The recombinant calreticulin constituted ˜32% of the solubleEscherichia coliproteins, and was purified to apparent homogeneity by ion exchange and hydrophobic liquid chromatography. As does thebona fideprotein, the recombinant calreticulin binds calcium and undergoes changes in its conformation upon Zn2+binding. We take this as a strong indication that the folding of theE.coli-expressed calreticulin is very similar, if not identical, to that of the authentic protein. Moreover, the bacterially expressed calreticulin readily reacted with anti-human and anti-rabbit antibodies, and the anti-recombinant calreticulin antibodies immunoreacted with HeLa calreticulin. The availability of this expression system will allow us to carry out site-specific and deletion mutagenesis analysis in structure-function studies of calreticulin.