自斷胜肽在蛋白質製備上的應用

Abstract

帶有親和性標誌的融合蛋白除了可增加溶解度，還能方便純化，但為了不影響目標蛋白的活性及後續用途，必須將標誌切除。為了方便切除標誌，於設計融合蛋白時即會插入一段連接胜肽，以符合蛋白酶的辨識序列。然而，蛋白酶價格不斐且切割後需要再次純化移除蛋白酶及切割下來的片段，而每次純化都會降低產率。 為了省去切割標誌的步驟，本研究在大腸桿菌中表現以可誘導自斷裂連接胜肽連接的親和性標誌與目標蛋白：H12S6-GFP-(EAAAR)2-RFP、MBP-H6-(EAAAK)2-InLα及MBP-H6-(EAAAK)2-G_CSF。調整溫度、pH值及時間，發現H12S6-GFP-(EAAAR)2-RFP在25℃、pH 8緩衝環境下，經過16-24小時可斷裂；MBP-H6-(EAAAK)2-InLα在室溫、pH 6緩衝環境下，經過42-48小時可斷裂；MBP-H6-(EAAAK)2-G_CSF在室溫、pH 6緩衝環境下，經過24-42小時可斷裂。並以H12S6-GFP-(EAAAR)2-RFP應用於「一鍋化純化」，先以親和性鎳管柱捕捉融合蛋白，於25℃、pH 8緩衝環境，經過24小時即可在上清液得到不具有親和性標誌與連接胜肽的RFP。

Affinity-tagged strategy to protein expression may increase the protein solubility and also enhance the ease of protein purification. Yet, for further application and to prevent protein of interest from losing biological activity, the tag has to remove before use. For this purpose, it is common to insert a peptide sequence with protease recognition site between the tag and target protein at the DNA manipulation stage. In addition to the cost of protease, the proteolytic treatment of a recombinant protein requires an extra effort to remove the unwanted fragments or protease. In this study, an autocleavable peptide was used to link affinity tag and protein of interest. Several recombinant proteins were prepared for this study as follows: H12S6-GFP-(EAAAR)2-RFP, MBP-H6-(EAAAK)2-InLα and MBP-H6-(EAAAK)2-G_CSF. Those proteins were overexpressed in E. Coli. A time-course monitoring of the autocleavage reaction of recombinant proteins at room temperature revealed that H12S6-GFP-(EAAAR)2-RFP, MBP-H6-(EAAAK)2-InLα and MBP-H6-(EAAAK)2-G_CSF could finish at pH8.0, 24 hs, pH6.0, 48 hr, and pH6.0, 42 hr, respectively. Furthermore, the application of “ one-pot purification” with H12S6-GFP-(EAAAR)2-RFP as an example were successfully demonstrated.