Du Dongning, Kato Tatsuya, Suzuki Fumiaki, Park Enoch Y
Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, Japan.
Mol Biotechnol. 2009 Oct;43(2):154-61. doi: 10.1007/s12033-009-9183-7. Epub 2009 Jun 7.
Three forms of recombinant protein complexes comprising the human prorenin (hPro) and (pro)renin receptor (hPRR) (hPRR/prorenin) were successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmids. They were localized in the fat body cells and formed a prorenin-bound hPRR complex. The expressed levels of hPro and hPRR were similar judging from Western blotting. The hPRR/prorenin complex containing 40 mug of hPRR (yield, 43%) and 30 mug of hPro (yield, 34%) was purified from 15 silkworm larvae by a series of purification using anti-FLAG and Strep-Tactin affinity chromatography. The renin activity of the purified hPRR/prorenin complex was 3.8-fold that of the mixture of hPRR and hPro expressed individually in vitro judging from the renin assay. These results show that the unstable transmembrane protein, hPRR, was coexpressed stably with ligand, hPro, and formed a stable protein, hPRR/prorenin complex that showed a high catalytic active form.
利用家蚕核型多角体病毒（BmNPV）杆粒在蚕幼虫中成功表达了三种包含人肾素原（hPro）和（前）肾素受体（hPRR）的重组蛋白复合物（hPRR/肾素原）。它们定位于脂肪体细胞中，并形成了与肾素原结合的hPRR复合物。从蛋白质印迹法判断，hPro和hPRR的表达水平相似。通过使用抗FLAG和链霉亲和素亲和层析的一系列纯化步骤，从15只蚕幼虫中纯化出了含有40μg hPRR（产率43%）和30μg hPro（产率34%）的hPRR/肾素原复合物。从肾素测定结果判断，纯化后的hPRR/肾素原复合物的肾素活性是体外单独表达的hPRR和hPro混合物的3.8倍。这些结果表明，不稳定的跨膜蛋白hPRR与配体hPro稳定共表达，并形成了一种稳定的蛋白——具有高催化活性形式的hPRR/肾素原复合物。
