马传染性贫血病毒Tat蛋白的生物活性及细胞内定位

Biological activity and intracellular location of the Tat protein of equine infectious anemia virus.

作者信息

Rosin-Arbesfeld R, Mashiah P, Willbold D, Rosch P, Tronick S R, Yaniv A, Gazit A

机构信息

Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel.

出版信息

Gene. 1994 Dec 15;150(2):307-11. doi: 10.1016/0378-1119(94)90443-x.

DOI:10.1016/0378-1119(94)90443-x

PMID:7821797

Abstract

The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10(11) bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by 'scrape loading'. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.

摘要

利用含有His.Tag前导序列的可诱导表达质粒pET16b在大肠杆菌中合成马传染性贫血病毒（EIAV）的Tat蛋白，从而能够通过金属亲和层析快速有效地富集带组氨酸标签的蛋白。从10¹¹个细菌细胞中可获得高达20mg的Tat产量。通过“刮取加载”将重组Tat蛋白导入犬类细胞后，显示其能有效反式激活EIAV长末端重复序列（LTR）。与HIV-1 Tat不同，发现EIAV Tat蛋白主要定位于细胞质中。大量纯化的功能性EIAV Tat蛋白的可得性应极大地促进详细的结构-功能分析。