Folding of the multidomain human immunodeficiency virus type-I integrase.
Folding of the multidomain human immunodeficiency virus type-I integrase.
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多域人类免疫缺陷病毒 I 型整合酶的折叠。
DOI：
10.1002/pro.5560030604
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Goodarzi,G
中科院分区：
文献类型：
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作者：
Grandgenett,DP;Goodarzi,G
从纯化的细菌包涵体中获得了人免疫缺陷病毒整合酶（IN），并建立了IN的蛋白折叠条件。IN用6 M胍· HCl-5 mM二硫苏糖醇变性，通过凝胶过滤纯化，并通过硫酸铵沉淀。用6 M盐酸胍可逆溶剂化沉淀的IN，使折叠反应中蛋白质浓度变化范围很大。用1 M NaCl缓冲液对变性IN进行6倍稀释，然后透析，产生具有酶活性的IN，能够使用各种人类免疫缺陷病毒-1（HIV-1）长末端重复DNA底物进行3′ OH末端加工、链转移和崩解。这些酶促反应的折叠IN制剂的比活性与直接从细菌中纯化的可溶性IN的比活性相当。折叠条件影响IN的亚基组成和酶活性。定义了标准折叠条件，其中产生了沉降为二聚体和四聚体的单体和蛋白质聚集体。这些蛋白质聚集体具有酶活性，而单体具有降低的链转移活性。折叠条件的温度改变允许主要形成单体。在测定时，这些单体对于链转移和崩解是有效的，但是在测定条件下IN的寡聚状态是不确定的。我们的研究结果表明，多结构域HIV-1 IN的单体正确折叠，具有各种催化活性，但在没有催化活性的情况下，特异性寡聚化的条件尚未确定。
Protein folding conditions were established for human immunodeficiency virus integrase (IN) obtained from purified bacterial inclusion bodies. IN was denatured by 6 M guanidine · HCl–5 mM dithiothreitol, purified by gel filtration, and precipitated by ammonium sulfate. The reversible solvation of precipitated IN by 6 M guanidine · HCl allowed for wide variation of protein concentration in the folding reaction. A 6‐fold dilution of denatured IN by 1 M NaCl buffer followed by dialysis produced enzymatically active IN capable of 3′ OH end processing, strand transfer, and disintegration using various human immunodeficiency virus‐1 (HIV‐1) long terminal repeat DNA substrates. The specific activities of folded IN preparations for these enzymatic reactions were comparable to those of soluble IN purified directly from bacteria. The subunit composition and enzymatic activities of IN were affected by the folding conditions. Standard folding conditions were defined in which monomers and protein aggregates sedimenting as dimers and tetramers were produced. These protein aggregates were enzymatically active, whereas monomers had reduced strand transfer activity. Temperature modifications of the folding conditions permitted formation of mainly monomers. Upon assaying, these monomers were efficient for strand transfer and disintegration, but the oligomeric state of IN under the conditions of the assay is indeterminate. Our results suggest that monomers of the multidomain HIV‐1 IN are folded correctly for various catalytic activities, but the conditions for specific oligomerization in the absence of catalytic activity are undefined.