DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis.
DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis.
复制标题
DNA 和肝素伴侣对来自亚马逊利什曼原虫的纯化重组复制蛋白 A 亚基 1 进行重折叠。
DOI：
10.1016/j.bbagen.2008.10.011
复制
发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Cano,MIN
中科院分区：
文献类型：
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作者：
Lira,CBB;Gui,KE;Perez,AM;daSilveira,RCV;Gava,LM;Ramos,CHI;Cano,MIN
复制蛋白A（RPA）是一种单链DNA结合蛋白，参与DNA代谢和端粒维持。来自亚马逊利什曼原虫的RPA亚基1（LaRPA-1）先前已经在含有富含G的端粒DNA的柱上亲和纯化。LaRPA-1在体内与寄生虫端粒结合并共定位。在这里，我们描述了天然重组LaRPA-1（rLaRPA-1）的纯化和表征。蛋白质最初通过使用尿素从包涵体中再溶解。透析后，rLaRPA-1可溶，但被DNA污染，通过溶解在尿素中的蛋白质的阴离子交换层析将其去除。然而，rLaRPA-1在透析去除尿素后沉淀。为了研究污染的DNA是否参与了rLaRPA-1的重折叠，在透析前将鲑鱼精子DNA或肝素加入溶液中。添加这些物质中的任一种都防止了rLaRPA-1的沉淀。得到的rLaRPA-1是可溶的，正确折叠，并能够结合端粒DNA。这是第一份报告显示的rLaRPA 1的特性和添加剂的重要性，在陪伴这种蛋白质的重折叠。rLaRPA-1的可用性应该有助于评估这种蛋白作为潜在药物靶点的重要性。
Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.