斑馬魚14-3-3蛋白家族的純化與性質鑑定

Abstract

14-3-3蛋白家族在真核生物中具有高度保守性，為大小約30kDa的酸性蛋白質。目前研究已知有兩百種以上的磷酸化蛋白能與14-3-3家族蛋白結合。這些蛋白幾乎參與細胞內所有的活動，如細胞週期、訊號傳遞、細胞凋亡等。斑馬魚已知的14-3-3家族共有十一種，實驗室已成功選殖其中九種，論文中將探討斑馬魚14-3-3家族蛋白的生化特性，首先利用大腸桿菌系統大量表現並純化帶有GST的斑馬魚14-3-3家族融合蛋白和帶有六個His的斑馬魚14-3-3家族融合蛋白。將已純化帶有六個His的14-3-3-β2、ζ1、ζ2、ε1及η蛋白，注射至兔子體內，以產生多株抗體，並分析抗體特性。根據實驗室先前實驗結果，在斑馬魚胚胎時期，過量表現14-3-3-ζ1於心臟，可觀察到斑馬魚心臟出現心律不整或心跳減緩的現象。因此利用與斑馬魚相似性很高的鯉魚之心臟萃取物，與斑馬魚14-3-3-ζ1進行GST pull down，並使用液相層析與串聯質譜儀比對斑馬魚蛋白資料庫進行分析，辨認出共十四種蛋白，其中包含β-actin-1。進一步驗證β-actin-1與14-3-3-ζ1的結合作用，利用GST pull down實驗發現在試管中β-actin-1與14-3-3-ζ1確實有結合反應。而將β-actin-1與14-3-3-ζ1轉染至COS-1細胞中，進行共同免疫沉澱，兩蛋白的結合反應依然存在。證實了斑馬魚β-actin-1與14-3-3-ζ1在試管中與細胞中皆有交互作用。我們利用相同的方法，驗證九種14-3-3家族蛋白的成員與β-actin-1的交互作用，透過GST pull down可觀察到在試管中，九種14-3-3家族蛋白皆與β-actin-1產生結合作用；而在細胞中，僅能發現ζ1、ζ2以及η這三個14-3-3家族成員，與β-actin-1有較強的結合反應。

The 14-3-3 proteins form a family of highly conserved acidic proteins in all eukaryotic cells with a subunit mass of approximately 30 kDa. 14-3-3 proteins were capable of interacting with more than 200 different phosphorylated proteins. The binding partners are involved in almost every cellular process, like cell cycle control, apoptosis and signal transduction. The zebrafish 14-3-3 gene family consists of 11 distinct 14-3-3 genes. Previously, our laboratory had cloned 9 members of the zebrafish 14-3-3 gene family. In our study, we have expressed and purified GST fusion proteins and His-tagged fusion proteins of the 9 zebrafish 14-3-3 protein from E.coli. His-tagged 14-3-3 recombinant proteins were then injected into rabbit to obtain polyclonal antibodies against 14-3-3 β2, ζ1 ,ζ2 ,ε1 and η; these antibodies were also characterized. Proteins pull downed by GST fusion 14-3-3-ζ1 in the carp’s heart extracts were analyzed to identify potential interacting partners of 14-3-3-ζ1. 14 proteins, including β-actin-1, were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the 14-3-3-ζ1 GST pull downed proteins. The interaction between 14-3-3-ζ1 and β-actin-1 was further confirmed by GST pull down and co-immunoprecipitation in COS-1 cells. Our results indicate that β-actin-1 is a binding partner of 14-3-3-ζ1 both in vitro and COS-1 cells. Furthermore, the interaction between other 14-3-3 isoforms and β-actin-1 were also analyzed. We demonstrated that all 14-3-3 family proteins are able to bind β-actin-1in the GST pull down assay in vitro. However, only three 14-3-3 isoforms including ζ1, ζ2 and η have the ability to interact with β-actin-1 in COS-1 cells.