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.

我们的第一个尝试是结晶杆菌病毒 在与TGF-B1复合物中表达的II型TGF-B受体。 由于受体上广泛的糖基化，结晶 使用该受体的试验未屈服于任何晶体。随后的 去糖基化过程产生了部分脱糖基化的 当与 TGF-B1。但是，这些晶体在X射线束中衍射很差 并且不适合确定结构。克服 碳水化合物异质性和低表达产率 杆状病毒表达系统，我们克隆了可溶性II 受体基因进入细菌表达载体。由于很大 该受体中存在的二硫键（12胱氨酸）的数量， 几个小组以前尝试重建受体 都失败了。我们的初步表达数据表明 大约20％的蛋白质在非聚集中表达 可溶性形式，其余形式以包容体形式表达 使用细菌系统。可溶部分显示出 ELISA分析的TGF-B结合。体外重构后， 从包含体材料中重折叠的受体也有效 ELISA分析。我们正在完善II型受体细菌 表达系统获得大量的受体蛋白 晶体学研究。