Hou Esther W, Prasad Rajendra, Beard William A, Wilson Samuel H
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Protein Expr Purif. 2004 Mar;34(1):75-86. doi: 10.1016/j.pep.2003.10.018.
We have devised simplified protocols to purify large quantities of histidine-tagged (His-tagged) and untagged heterodimeric forms of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). Here, we report the optimization of overexpression and purification of heterodimeric RT expressed in Escherichia coli. The coding sequences of p66 and p51 subunits of RT were amplified using PCR from HXB2 HIV-1 and cloned into a bacterial expression system. The resulting expression plasmids for the RT subunits, pET-RT66 and pET-RT51, were under a strong T7/lac promoter that is induced by isopropyl-beta-d-thiogalactopyranoside. Purification of heterodimeric forms of RT was facilitated by high-level expression of these subunits that represented approximately 30-40% of total cell protein. For purification of the His-tagged heterodimeric RT, cell pellet from cells expressing the untagged p66 subunit was mixed in excess with a cell pellet expressing tagged p51. For untagged heterodimeric RT, the pellet from cells expressing p51 was mixed in excess with pellet expressing p66. Subunit dimerization occurred during cell lysis. During the subsequent chromatography steps, stable p66/p51 heterodimer was purified to homogeneity. The heterodimeric nature of the final preparations of RT was confirmed by analytical gel filtration, mass spectrometry, and denaturing gel electrophoresis. Further, the sensitivity of these enzyme preparations to AZTTP indicated that the histidine tag had no effect on nucleoside inhibitor binding, nucleotide binding or insertion, or DNA binding. The application of these expression/purification methodologies represents a useful method to purify large quantities of heterodimeric RT for structural investigations and provides an efficient protocol to produce subunit-specific amino acid alterations necessary for unambiguous structure/function investigations.
我们设计了简化方案，用于大量纯化人免疫缺陷病毒1型逆转录酶（HIV-1 RT）的组氨酸标签化（His标签化）和无标签异二聚体形式。在此，我们报告了在大肠杆菌中表达的异二聚体RT的过表达和纯化的优化情况。使用PCR从HXB2 HIV-1扩增RT的p66和p51亚基的编码序列，并克隆到细菌表达系统中。所得的RT亚基表达质粒pET-RT66和pET-RT51，受异丙基-β-D-硫代半乳糖苷诱导的强T7/lac启动子控制。这些亚基的高水平表达促进了RT异二聚体形式的纯化，其表达量约占总细胞蛋白的30%-40%。为了纯化His标签化的异二聚体RT，将表达无标签p66亚基的细胞沉淀与过量表达标签化p51的细胞沉淀混合。对于无标签异二聚体RT，将表达p51的细胞沉淀与过量表达p66的沉淀混合。亚基二聚化在细胞裂解过程中发生。在随后的色谱步骤中，稳定的p66/p51异二聚体被纯化至同质。通过分析凝胶过滤、质谱和变性凝胶电泳证实了RT最终制剂的异二聚体性质。此外，这些酶制剂对AZTTP的敏感性表明，组氨酸标签对核苷抑制剂结合、核苷酸结合或插入或DNA结合没有影响。这些表达/纯化方法的应用代表了一种用于大量纯化异二聚体RT以进行结构研究的有用方法，并提供了一种有效的方案来产生明确的结构/功能研究所必需的亚基特异性氨基酸改变。
