Ozturk D H, Erickson-Viitanen S
Virology Department, The DuPont Merck Pharmaceutical Company, Experimental Station, Building E336/1B, Wilmington, Delaware, 19880-0336, USA.
Protein Expr Purif. 1998 Oct;14(1):54-64. doi: 10.1006/prep.1998.0934.
An efficient method for the expression and purification of nucleocapsid precursor protein (p15NC) from HIV-I (BH 10 isolate) was developed and used to obtain large quantities of this viral protein for structural studies, protein biochemistry, and high-throughput screening efforts. We have engineered an existing p15NC clone into a new vector developed at the University of Heidelberg, Germany. Using PCR, we introduced new restriction sites and a strong ribosome-binding site in the p15NC gene and expressed authentic p15NC protein. Our protocol enabled us to rapidly obtain soluble and highly stable p15NC expressed in Escherichia coli and to purify several milligrams of p15NC to homogeneity. In the current purification scheme, lysis of cell paste followed by a simple three-step FPLC procedure yields about 0.4-0.5 mg of purified p15NC per gram of E. coli cell paste expressing the protein with an overall yield of 45%. The purified p15NC retained its ability to bind full-length HIV-I p15NC mRNA in solution- or solid-phase-based assays. A specific stem-loop forming RNA fragment (24-mer) and its antisense DNA oligomer (21-mer) derived from the full-length p15NC mRNA were also able to bind to p15NC. In addition, antisense DNA oligos with bulky 5-iodouracil and 5-iodocytidine substituents were able to bind to p15NC with little or no perturbations as assessed by their ability to compete with the full-length p15NC mRNA in filter-binding competition assays. In addition, RNA-dependent cleavage of the purified p15NC in vitro by HIV-I protease occurred at rates similar to those reported previously.
开发了一种从HIV-1（BH10分离株）中表达和纯化核衣壳前体蛋白（p15NC）的有效方法，并用于获得大量该病毒蛋白，以进行结构研究、蛋白质生物化学和高通量筛选工作。我们已将现有的p15NC克隆构建到德国海德堡大学开发的一种新载体中。通过聚合酶链反应（PCR），我们在p15NC基因中引入了新的限制性酶切位点和一个强核糖体结合位点，并表达出了天然的p15NC蛋白。我们的方案使我们能够快速获得在大肠杆菌中表达的可溶性且高度稳定的p15NC，并纯化出几毫克均一的p15NC。在当前的纯化方案中，细胞糊状物裂解后通过简单的三步快速蛋白质液相色谱（FPLC）程序，每克表达该蛋白的大肠杆菌细胞糊状物可产生约0.4 - 0.5毫克纯化的p15NC，总产率为45%。纯化后的p15NC在基于溶液或固相的检测中保留了其与全长HIV-1 p15NC mRNA结合的能力。一个源自全长p15NC mRNA的特定茎环形成RNA片段（24聚体）及其反义DNA寡聚体（21聚体）也能够与p15NC结合。此外，可以通过在滤膜结合竞争检测中与全长p15NC mRNA竞争的能力评估，带有大体积5 - 碘尿嘧啶和5 - 碘胞嘧啶取代基的反义DNA寡聚体能够与p15NC结合，且几乎没有或没有干扰。此外，HIV-1蛋白酶在体外对纯化的p15NC进行RNA依赖性切割的速率与先前报道的相似。
