Structure Determination of TGF-beta Receptor

TGF-β受体的结构测定

• 批准号: 6099133
• 负责人: PETER D. SUN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6099133
• 关键词: Baculoviridae; crystallization; disulfide bond; enzyme linked immunosorbent assay; glycosylation; growth factor receptors; molecular cloning; protein folding; protein reconstitution; protein structure; receptor expression; structural biology; transfection /expression vector; transforming growth factors

Our first attempt was to crystallize a baculovirus expressed human type II TGF-b receptor in complex with TGF-b1. Due to extensive glycosylation on the receptor, the crystallization trials with this receptor did not yield to any crystals. Subsequent deglycosylation procedure yielded a partially deglycosylated receptor which produced small crystals when complexed with TGF-b1.However, these crystals diffract poorly in the X-ray beam and not suitable for structure determination. To overcome the carbohydrate heterogeneity and low expression yield of the baculovirus expression system, we have cloned the soluble type II receptor gene into a bacteria expression vector. Due to a large number of disulfide bonds (12 cystines) present in this receptor, previous attempts by several groups to reconstitute the receptor have all failed. Our preliminary expression data suggests that approximately 20% of the protein is expressed in a non-aggregated soluble form and the rest is expressed in an inclusion body form using the bacteria system. The soluble part is shown to posses TGF-b binding by ELISA assays. After in vitro reconstitution, the refolded receptor from the inclusion body material is also active in the ELISA assay. We are refining the type II receptor bacteria expression system to obtain large quantity of receptor protein for crystallographic studies.

我们的第一次尝试是使杆状病毒结晶 表达与 TGF-b1 复合的人 II 型 TGF-b 受体。 由于受体上广泛的糖基化，结晶 对这种受体的试验没有产生任何晶体。随后的 去糖基化过程产生了部分去糖基化的 与受体复合时产生小晶体 TGF-b1。然而，这些晶体在 X 射线束中的衍射性能很差 且不适合结构测定。为了克服 碳水化合物异质性和低表达产量 杆状病毒表达系统，我们克隆了可溶性II型 受体基因转化为细菌表达载体。由于大 该受体中存在的二硫键数量（12 个胱氨酸）， 之前几个小组尝试重建受体 都失败了。我们的初步表达数据表明 大约 20% 的蛋白质以非聚集形式表达 可溶形式，其余以包涵体形式表达 使用细菌系统。显示可溶部分具有 通过 ELISA 测定 TGF-b 结合。体外重构后， 来自包涵体材料的重折叠受体也具有活性 ELISA 测定。我们正在精炼II型受体细菌 表达系统以获得大量的受体蛋白 晶体学研究。