Selective isolation and purification of Tat protein via affinity membrane separation
Selective isolation and purification of Tat protein via affinity membrane separation
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DOI：
10.1021/bp049804z
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发表时间：
2005-03-01
影响因子：
2.9
通讯作者：
Bhattacharyya, D
中科院分区：
文献类型：
--
作者：
Hollman, AM;Christian, DA;Bhattacharyya, D
本工作涉及利用亲和膜系统从复杂发酵液中分离达特蛋白。达特是一种调节蛋白，对HIV-1复制至关重要，因此是疫苗和药物开发的潜在候选者。此外，达特可以促进外源分子跨细胞膜的转运，并与HIV痴呆的发病机制有关。亲和膜通过在4-堆叠膜构造内偶联抗生物素蛋白来制备。细菌裂解物进料中的达特（天然生物素化）可及性受RNA酶、蛋白质浓度和离子强度的影响。增强的可访问性转化为每次通过的整体产品产量的显着增加。通过SDS-PAGE、Western印迹、活性测定和神经毒性研究，将膜分离的达特的纯度与通过填充柱色谱法制备的纯度进行比较。通过膜分离产生的达特蛋白主要产生单体形式的寡肽序列，而柱色谱主要产生聚合形式的达特。这些差异导致两种制剂的神经毒性和细胞摄取发生变化。
This work deals with the separation of Tat protein from a complex fermentation broth using an affinity membrane system. Tat is a regulatory protein that is critical for HIV-1 replication and thus a potential candidate for vaccine and drug development. Furthermore, Tat can facilitate transport of exogenous molecules across cell membranes and is implicated in pathogenesis of HIV dementia. Affinity membranes were prepared through coupling of avidin within a 4-stack membrane construct. Tat (naturally biotinylated) accessibility in the bacterial lysate feed was influenced by the presence of RNAse, protein concentration, and ionic strength. Enhanced accessibility translated to a marked increase in the overall product yield per pass. The purity of the membrane-isolated Tat was compared to that prepared via packed column chromatography through SDS-PAGE, Western blot, activity assay, and neurotoxicity studies. Tat protein produced via membrane separation yielded primarily monomeric forms of the oligopeptide sequence, whereas column chromatography produced predominately polymeric forms of Tat. These differences resulted in changes in the neurotoxicity and cellular uptake of the two preparations.