Expression of mRNA cap-binding protein and structure analysis of its cap recognition mechanism

帽结合蛋白(CBP或IF-4E)在启动核糖体蛋白质合成中起着重要作用，在活细胞中含量很低。为了阐明该蛋白的生化和结构性质，以及对mRNA帽结构的选择性识别，我们开展了这项研究。(1)成功地合成了全长660个碱基对的人帽结合蛋白(HCBP)基因。(2)将hCBP基因整合到pBR322载体中，实现了hCBP与人生长激素的包涵体的高效表达。(3)建立了hCBP的分离纯化方法。(4)对7-甲基鸟嘌呤碱基(M7G)所表征的mRNA帽结构可能具有选择性识别功能的氨基酸残基进行了研究。模型交互作用系列研究。(5)基于以上洞察，通过定点突变，将hCBP的功能氨基酸转化为非功能氨基酸。建立了基因突变和突变型hCBP蛋白的分离纯化方法。(6)化学合成了一系列mRNA帽类似物。(7)用紫外光谱和荧光光谱研究了这些帽类似物与天然和突变型hCBP蛋白的相互作用模式。结果表明，His-33、His-37、Trp-102和Glu-105残基在与mRNA帽子结构的相互作用中起着重要作用。此外，含有m7G的柱与天然和突变的hCBPs的亲和力实验也得到了相同的结果。(8)X射线晶体和核磁共振溶液分析正在进行中。

Cap-binding protein (named CBP or IF-4E), the content of which is very small amount in living cell, plays an important role in initiating the protein synthesys on ribosome. In order to elucidate the biochemical and structural nature of this protein and to make clear the selective recognition of mRNA cap structure, we carried out this research project. The main results obtained so far are as follows:(1) We succeeded in the chemical synthsis of gene of human cap-binding protein (hCBP)(total base pairs of 660).(2) We established the inclusion of this gene into pBR322 vector and the large amount of expression of hCBP as the inclusion body with human growth hormone.(3) We established the separation of hCBP from the confused protein and the HPLC purification method.(4) The functional amino acid residues which probably functions in the selective recognition for the mRNA cap structure characterized by 7-methylguanine base (m7G) were surveyed by a series of model interaction studies.(5) Based on the above insight, the functional amino acids of hCBP were transformed to the non-functional amino acids by the site-directed mutagenesis. The methods for the gene mutations and the isolation and purification of mutant hCBP proteins were established.(6) A series of mRNA cap analogues were chemically synthesized.(7) The interaction modes of these cap analogues with the native and mutant hCBP proteins were investigated by UV and fluorescence spectroscopies. As a result, it was made clear that the His-33, His-37, Trp-102 and Glu-105 residues play important role in interaction with mRNA cap structure. Further, the same results were obtained from the binding experiments of the affinity of m7G-containing column with native and mutant hCBPs.(8) The X-ray crystal and NMR solution analyses are now in progress.