新型海洋生物色澤蛋白之基因、結構與產生不同成色的突變和機制

Molecular Cloning, Structural Characterization and Color-regulating Mechanism of Novel Chromoprotein Isolated from Marine Organisms

江政一(Cheng-Yi Chiang)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/分子與細胞生物學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.01084

摘要

GFP-like protein family在生物學應用上為良好的marker以及reporter，傳統螢光蛋白常需利用螢光設備幫助觀察因而具有限制性，且色彩主要侷限於紅、黃、綠等顏色。為了拓展可應用的色彩，以及提高觀察的便利性，我們希望獲取新種的藍色或紫色色澤蛋白。我們從表現天然藍、紫色色彩的地毯海葵觸手組織中選殖出新種的紫色色澤蛋白shCP與新種的藍色色澤蛋白sgBP。純化後的shCP於574 nm處有最大的吸收峰值，sgBP於608 nm處有最大的吸收峰值。藉由結構導向的定點突變法改變色澤蛋白chromophore以及chromophore周圍5 Å的氨基酸殘基，發現對於shCP chromophore的第63位置的氨基酸glutamate突變為serine(E63S)時，可使原來的紫色蛋白呈現粉紅色；而蛋白質序列具Q39S突變時，則為紅色；另一方面，位於sgBP chromophore周邊第157位置的serine突變為S157C時，可將突變蛋白變為深藍色色澤。為了證明shCP與sgBP做為生物性標記(biomarker)的實用性，我們構築好以斑馬魚α-actin啟動子表現shCP、sgBP與及其突變型色澤蛋白的表現載體並以顯微注射法進行基因轉殖。經轉殖後的胚胎在飼育至性成熟後可篩選出具有transient色澤表現的成魚，例如帶有shCP表現載體的斑馬魚其肌肉中會出現紫紅色；帶有shCP-E63S的斑馬魚可呈現粉紅色；帶有sgBP的斑馬魚則可表現藍色，且由配對實驗可確認色澤基因可被穩定遺傳至子代。本研究應用新種色澤蛋白並找出可利用突變造成chromophore deprotonation的效果進而影響蛋白顏色表現的機制，並且確認色澤蛋白與其突變衍生物做為生物性標記的應用性。

關鍵字

色澤蛋白 ； 生物標記 ； 蛋白質工程 ； 基因轉殖

並列摘要

GFP-like proteins have been studied with the aim of developing fluorescent proteins. Since the property of color variation is understudied, we isolated novel GFP-like chromoproteins from the carpet anemone Stichodactyla haddoni and Stichodactyla gigantea, termed shCP and sgBP, respectively. The shCP protein has maximum absorption wavelength peak (λmax) is located at 574 nm, resulting in a purple appearance. The sgBP has a λmax at 608 nm and shows a blue appearance. We mutated aa residues to enhance the color properties according to the simulated structure of shCP and the crystal structure of sgBP. With the mutations in and surrounding 5 Å of the chromophore, we found that the mutated proteins exhibited altered optical properties. The λmax of chromophore-mutated protein shCP-E63S was shifted to 560 nm and exhibited a pink color. shCP with surrounding mutations Q39S and T194I also exhibited coloration changes to red and purple-blue. An additional mutation at I196H of the mutated protein shCP-E63L exhibited green fluorescence. In the chromoprotein sgBP, the surrounding S157C mutation shifted the λmax to 604 nm and darkened the blue color expression, indicating the color expression of chromoprotein could be altered by deprotonation state change of the phenolic group in the chromophore. Additionally, we found that the cDNAs of shCP, sgBP and its mutated varieties are faithfully and stably expressed both in Escherichia coli and zebrafish embryos. Our results provide a structural basis for the color regulation of the biomarker development.

並列關鍵字

chomoprotein ； biomarker ； protein engineering, transgenesis

參考文獻

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