Glycoforms of Phaseolin-Structure, Targeting and Stability

我们建议定位可能负责种子贮藏蛋白小菜豆的靶标指定和分类的蛋白质决定因素。这将通过在克隆的小菜豆基因编码序列中产生缺失和构建小菜豆/GUS基因嵌合体来实现，随后将利用钛质粒介导的转化技术将其转化到烟草中。然后将对转基因种子中不同结构的表达和定位进行分析，以建立特定多肽序列和靶向特性之间的关联。还将在感染杆状病毒表达载体的昆虫细胞系中研究N-连接的糖基化和小菜豆的分选。我们希望最终能够将分子多肽部分的特定序列和寡糖的功能与它们的结构联系起来。在另一项提议中考虑的研究中，小红豆素原构体的三级结构将通过X射线结晶学来确定。

We propose to map the protein determinants which are likely to be responsible for target specification and sorting of the seed storage protein phaseolin. This will be approached by producing deletions in the coding sequence of a cloned phaseolin gene and by constructing phaseolin/GUS gene chimeras, which will be subsequently transferred into tobacco by using Ti plasmid- mediated transformation techniques. Analysis of the expression and localization of the different constructs in transgenic seeds will then be performed, to establish a correlation between specific peptide sequences and targeting properties. N-linked glycosylation and sorting of phaseolin, will also be studied in an insect cell line infected with a baculovirus expression vector. We intend to eventually be able to relate the function of specific sequences on the polypeptide portion of the molecule and of oligosaccharides, to their structure. In studies contemplated in a separate proposal, the tertiary structure of the phaseolin protomers will be determined by X-ray crystallography.