單胞綠藻SUMO E1活化酶之基因選殖與蛋白質表現

Abstract

SUMO (small ubiquitin-like modifier) 是一種類泛素蛋白質。其可以藉由共價結合的方式接到目標蛋白質上，以進行轉譯後修飾作用。SUMO化修飾作用需經過一連串由SUMO E1活化酶、E2銜接酶、E3黏合酶所進行的酵素反應。E1活化酶是由SAE1 (sumo activating enzyme subunit 1) 與SAE2 (sumo activating enzyme subunit 2) 兩個次單元體所組成，並負責SUMO化的起始步驟。其功能為將SUMO活化並轉移至E2銜接酶上。 本論文先以酵母菌、人類及阿拉伯芥的SUMO E1活化酶序列進行BLAST分析，並利用AUGUSTUS軟體預測exon-intron找出可能為單胞綠藻 (Chlamydomonas reinhardtii) SUMO E1活化酶的序列。利用專一性引子，本論文成功選殖出SAE1次單元體及SAE2次單元體之部分序列。嘗試不同的表現與純化條件發現，在有GST (glutathione S-transferase) 協助摺疊的情況下，可得到正確表現之重組蛋白。而無論直接使用CrSAE2部分序列或加上CrSAE1和CrSAE2部分序列一起進行in vitro pull down實驗，皆沒有觀察到其與CrSUMO96-GG間之交互作用。本論文亦將表現情形較佳之GST-SAE2部分序列做為抗原，製備SAE2次單元體之多株抗體，以作為未來研究的工具。

SUMO (small ubiquitin-like modifier) is one of the ubiquitin-like proteins, which can covalently attach to target proteins for post-translational modification. SUMOylation cascade includes SUMO E1 activating enzyme, E2 conjugating enzyme and E3 ligases. SUMO E1 activating enzyme is a heterodimer composed of SAE1 (sumo activating enzyme subunit 1) and SAE2 (sumo activating enzyme subunit 2). I initially performed the BLAST search by using the SUMO E1 sequences of Saccharomyces cerevisiae, Arabidopsis thaliana and human as the template, and predicted the potential exon-intron regions by AUGUSTUS program to find out the potential E1 activating enzyme of Chlamydomonas reinhardtii. In this study, the SAE1 and SAE2 partial sequences were successfully cloned by using specific primers respectively. The results showed that GST (glutathione S-transferase) can help the protein expression of SAE1 and SAE2 in Escherichia coli. The in vitro pull down assay did not reveal an interaction between SAE2 partial sequence and CrSUMO96-GG. Furthermore, I also used the GST-CrSAE2 partial sequence as the antigen for preparing the polyclonal antibody. Hopefully, it can be the specific tool for study of C. reinhardtii SUMO E1 activating enzyme in the future.