泛素化修飾分析方法之再探討

Abstract

AtMAPRs 家族蛋白質包含 AtMAPR2、AtMAPR3、AtMAPR4 和 AtMAPR5。在 E1、E2、ATP 和泛素的環境中，GST-AtMAPR3 和 GST-AtMAPR5 的重組蛋白質可接上泛素 (林盈涵, 2009)。由於以上的實驗中沒有作 GST 的負控制組，因此以胞外泛素化修飾實驗分析 GST。經由免疫染色分析發現，GST (26 kD) 上方有一分子量上升之色帶 (35 kD)，此結果可推論GST 可能具有自身泛素化活性。進一步利用 pull down assay 分析 GST 與泛素可能之交互作用，並利用軟體模擬 GST 與泛素交互作用之模式。此外，利用胞外泛素化修飾實驗，分析不同長度之 His-AtMAPR3 (AtMAPR3, AtMAPR3ΔTM 和 AtMAPR3ΔTMΔC) 和 His-AtMAPR5 (AtMAPR5ΔTM 和 AtMAPR5ΔTMΔC)的自身泛素化活性，其中只有 His6-AtMAPR5ΔTM (70-220 a.a.) 觀察到可能具有胞外自身單一泛素化活性。GST是否會影響 His6-AtMAPR5ΔTM (70-220 a.a.) 的自身泛素化活性，還待進一步的實驗驗證。

In Arabidopsis, AtMAPR family consists of four members which are AtMAPR2, AtMAPR3, AtMAPR4 and AtMAPR5. In the presence of E1, E2, ATP and ubiquitin, recombinant proteins of GST-AtMAPR3 and GST-AtMAPR5 are modified by ubiquitin. To study the effect of GST on this reaction, purified GST protein was subjuected to in vitro ubiquitination assay. A shifted protein band of 35 kD in addition to the GST (26 kD) appeared on a immunoblot of SDS-PAGE stained with anti-GST antibody. This result suggested that GST alone may be subjected to autoubiquitination. Pull-down analysis was needed to study the possible interaction between GST and ubiquitin. Protein-protein docking was performed to simulate this interaction. On the other hand, the autoubiquitination activity of His-AtMAPR3 (AtMAPR3, AtMAPR3ΔTM and AtMAPR3ΔTMΔC) and His-AtMAPR5 (AtMAPR5ΔTM and AtMAPR5ΔTMΔC) were also examined by in vitro ubiquitination assay, but only His6-AtMAPR5ΔTM (70-220 a.a.) showed auto-monoubiquitinaiton activity. In order to clarify whether GST affects the auto-monoubiquitination activity of His6-AtMAPR5ΔTM (70-220 a.a.) or not, more experiments should be done in the future.