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標題: 酵母菌MATa1蛋白質的大量純化和長晶
Large Scale Purification and Crystallization of Yeast Mating Type MATa1 Homeodomain protein
作者: 陳靚珮
Chan, Ching-Pei
關鍵字: homeodomain;MATa1蛋白質;MATa1;crystallization
出版社: 生物化學研究所
全長為126個基酸的MATa1蛋白質是屬於homeodomain family的一員,在酵母菌a/a cell type中(酵母菌有a、a or a/a cell等3種cell types),a1和同為homeodomain蛋白質的MATa2,擔任著調控酵母菌偶配細胞組態(mating type)的角色。當MATa1蛋白質與主調控轉錄因子a2蛋白質形成二複合體後,能抑制單套染色體細胞(a or a cell)特有基因─ haploid-specific gene (hsg)的表達,使偶配後具有雙套染色體的cell type (a/a cell),呈現diploid cell的表徵。
依據許多研究資料得知,homeodomain蛋白質和特定DNA序列鍵結的主要作用區,為位於C-端一段含有60個基酸的保守序列。a1與a2和其他homeodomain蛋白質不同的是採用相互作用的機制來調控對DNA序列的選擇性。各別a1與a2蛋白質對DNA的親合力與選擇性很弱(a1:Km >10-5 M;a2:Km~10-8 M),但是當a1、a2形成二複合體,造成彼此空間結構的改變後,MATa1對特定DNA序列的鍵結及辨識能力則增加3000倍以上(a1/a2:7×10-17 M2)。
由於MATa2/DNA、MATa1/MATa2/DNA的晶體結構陸續被解析出來,促使我們想了解單獨a1蛋白質的三度空間結構,除了定出a1 N-端序列的結構位置外,更進一步想得知a1在形成heterodimer時,晶體結構上是產生了那些細微的改變,造成a1對DNA的親合力有很大的不同。
故本研究計劃以大腸桿菌為宿主系統,大量表達酵母菌mating type MATa1不同片段蛋白質─T43 (43-126)和K66 (66-126),釐定蛋白質純化步驟後,持續純化和濃縮 (濃度~24 mg/ml,total~ 65 mg,純度>99%)不同片段蛋白質,持續進行MATa1蛋白質長期結晶實驗,以為測定MATa1蛋白質結構的前期實驗。

In the yeast (Saccharomyces cerevisiae), the cell mating type (a, a or a/a cell) is determined at the transcription level through different combinatorial control of regulatory proteins, a2, a1 and MCM1. The a1/a2 homeodomain heterodimer, which binds to the operator sites upstream of the haploid cell type specific genes, deactivate the hsgs expression in a/a diploid cells. Physiologically, a/a cells are incapable of mating, a distinct property of the a and a haploid cells.
Both MATa1(126 amino acids) and MATa2(210 amino acids) are
members of homeodomain superfamily which play central role in cell developmental biology. Molecular biology studies show that homeodomain proteins contain a region of 60 conserved amino acids for DNA-binding. The DNA binding features of a1 and a2 proteins among others homeodomain proteins are that affinity and specificity of a1 and a2 proteins are mediated
through protein-protein interaction. The monomeric form of a1 and a2 protein have very low DNA-binding affinity, a1: Km > 10-5 M and a2: Km ~10-8 M respectively. Upon formation of a1/a2 heterodimer, the DNA binding affinity of MATa1 increases by a factor of at least 3000 fold (a1/a2: 7×10-17 M2).
The solved crystal structures of MATa2/DNA and MATa1/MATa2/DNA ternary complexes prompt us to investigate the structural detail of a1 protein alone. The goal is to understand the variations of a1 conformation in a1/a2 heterodimer formation and the N-terminal arm structure which is required for DNA binding in most homeodomain proteins but disorder in the MATa1/MATa2/DNA structure.
In this report, we over-express large quantify of a fragment of the a1 protein ─T43(43-126 amino acids) in E. Coli system. Then we purify and concentrate the protein (concentration ~24 mg/ml, total ~65 mg, purity > 99 %) for MATa1 protein crystallization experiment.
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