STRUCTURE DETERMINATION OF TGF-BETA RECEPTOR

TGF-β受体的结构测定

• 批准号: 6289000
• 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/6289000
• 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-beta receptor in complex with TGF-beta 1. Due to extensive glycosylation on the receptor, the crystallization trials with this receptor did not yield any crystals. Subsequent deglycosylation procedures yielded a partially deglycosylated receptor which produced small crystals when complexed with TGF- beta 1. However, these crystals diffract poorly in the X-ray beam and are 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 possess TGF-beta binding by ELISA assays. After in vitro reconstitution, the refolded receptor from the inclusion body material is also active in the ELISA assay. We have made several bacterial constructs with varying lengths in the N-terminal amino acid sequence of the type II TGF-beta receptor for crystallization. A similar construct corresponding to the full- length extracellular domain of the type I TGF-beta receptor was also made for protein expression and crystallization. We are refining the type I and II receptor bacteria expression systems to obtain large quantities of the receptor proteins for crystallographic studies. - TGF-beta receptor, crystal structure, protein expression, ELISA assay, cytokine

我们的首次尝试是在与TGF-β1中结晶杆状病毒表达的人II型TGF-β受体。由于受体上的广泛糖基化，该受体的结晶试验没有产生任何晶体。随后的去糖基化过程产生了部分脱脂的受体，当与TGF-β1复合时会产生小晶体。但是，这些晶体在X射线束中衍射较差，不适合结构测定。为了克服杆状病毒表达系统的碳水化合物异质性和低表达产率，我们将可溶性II型受体基因克隆到细菌表达载体中。由于该受体中存在的大量二硫键键（12胱氨酸），几个组以前尝试重建受体的尝试都失败了。我们的初步表达数据表明，大约20％的蛋白质以非聚集的可溶性形式表达，其余的使用细菌系统以包含体形式表达。可溶部分显示出通过ELISA分析具有TGF-β结合。体外重构后，来自包含体材料的重折叠受体在ELISA测定中也很活跃。我们制作了几种细菌构建体，在II型TGF-β受体的N端氨基酸序列中，用于结晶。也对应于I型TGF-β受体的全长细胞外结构域的类似构建体也用于蛋白质表达和结晶。我们正在完善I型和II受体细菌表达系统，以获得大量的受体蛋白进行晶体学研究。 -TGF-β受体，晶体结构，蛋白质表达，ELISA测定，细胞因子