人類類Ste20蛋白激酶Mst4的表現、純化和特性之研究

Abstract

Mst4 (哺乳類類Ste20蛋白激酶，Mammalian Ste20-like Kinase 4)是屬於人類類Ste20蛋白激酶(human Ste20-like kinase)中的胚胎中心激酶III家族(GCK-III，germinal center kinase)的一員。目前已知Mst4的生物功能卻具有爭議性。故Mst4生物特性之研究，對於釐清其角色與作用機轉是非常重要的。所以本研究採用大腸桿菌重組表現系統，大量表現並純化Mst4以利其相關特性之研究。 有趣的是，Mst4只在特定一株修飾過的菌株BL21(DE3)-RIL上有表現，而在另外兩株中則沒有。Mst4之截斷活化株Mst4Δ296在自我磷酸化後 (auto-phosphorylation )顯示了Mst4 具有磷酸化的酪胺酸殘基。另外，我們也採用蛋白質交互作用沉澱試驗(pull down assay)找出了可能與Mst4有交互作用的蛋白質，將利用介質輔助雷射脫附離游離-飛行時間式質譜儀(MALTI-TOF, matrix assisted laser desorption ionization-time of flight mass spectrometry)來加以分析，以利推測及繼續研究其相關調控機轉。

Mst4 (mammalian Ste20-like kinase 4) is a novel member of GCK- III (germinal center kinase) subfamily of human Ste20-like protein kinases. The biological function of Mst4 is unclear by far. Thus, it is important to investigate the biological properties of Mst4 to understand more about Mst4. In this study, we overexpressed recombinant Mst4 in E. coli and purified to studying Mst4. Interestingly, Mst4 could only expressed in a modified E. coli BL21 (DE3) strain BL21 (DE3)-RIL but not two other. Autophosphorylation of Mst4Δ296, a dominant active form of Mst4 showed that Mst4 contains phosphotyrosine residue, but not phosphothreonine. In addition, we tried to find out the Mst4-associated proteins by pull down assay. The associated proteins will be further identified by MALTI-TOF (matrix assisted laser desorption ionization-time of flight mass spectrometry). The identification of Mst4 associated proteins may provide major clued to understand the molecular mechanism of Mst4 dependent cellular responses.