Glycoforms of Phaseolin-Structure, Targeting and Stability

我们建议绘制蛋白质决定因素，这可能是负责目标规格和分类的种子储存蛋白phaseolin。这将通过在克隆的phaseolin基因的编码序列中产生缺失和构建phaseolin/GUS基因嵌合体来实现，这些嵌合体随后将通过Ti质粒介导的转化技术转移到烟草中。分析不同构建体在转基因种子中的表达和定位，建立特定肽序列与靶向特性之间的相关性。在感染杆状病毒表达载体的昆虫细胞系中，还将研究相蛋白的n -链糖基化和分选。我们打算最终能够将分子多肽部分和低聚糖上的特定序列的功能与它们的结构联系起来。在一项单独提案中考虑的研究中，phaseolin原相的三级结构将由x射线晶体学确定。

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.