以生物科技生產Haloarcula marismortui HmBRI蛋白質在生物工業上應用之研究

Abstract

中文摘要 細菌視紫紅質被發現在嗜鹽古細菌的紫膜 (purple membrane) 上，當嗜鹽古細菌遭遇到低氧氣以及高光照的環境，這樣的蛋白質就會被表現。其有七個穿膜 α 螺旋，並在蛋白質的中心以schiff base 形式連結 retinal 。接受光照時，蛋白質內部的發色基團 retinal 會產生形變，將氫離子從蛋白質的一端運輸至另一端，而在細胞膜的兩側形成一個氫離子的梯度。這樣的化學能梯度可藉由 ATP synthase 而達到 ADP 的磷酸化，以讓嗜鹽古細菌能在氧氣量較低的環境產生能量。細菌視紫紅質由於內部有一個發色基團，因此擁有特殊吸收光譜，光週期；又因為其在原物種內的高產量及穩定性，讓他成為一個很有潛力的生物感光材料。本實驗藉由電腦的序列分析，以及光譜測定，光週期測定和氫離子幫浦能力來測試 HmBRI ( Haloarcula marismortui BRI ) 的性質，確認其為細菌視紫質；並且發現另外幾個具有點突變之 HmBRI，其中 D94N 這個突變可以讓 HmBRI 在 E.coli 系統內有極高的表現量，且其對於熱和光穩定，也可處於無水環境下仍保有其特性。在定性測試後，本實驗也進行了初步的應用測試，發現以 E.coli 系統所表達之 HmBRI 和其突變蛋白質，有很高的生物材料應用潛力。

Abstract Bacteriorhodopsin (BR) is a protein exists in the purple membrane of some archaea and it is expressed when environment is illuminated with strong light or is low in oxygen. BR consists of seven trans-membrane α-helix with a retinal in the core as a chromophore that form schiff base with a Lys residue on the helix G. In the presence of light illumination, protein conformational will change due to the isomerization of retinal, and it further leads to an outward proton transport to generate a proton gradient across membrane. With this chemical gradient, ATP synthase generates energy under low oxygen condition. Just like other photoreceptors, BR features a maximum absorption wavelength and a millisecond time scale photocycle. The fact that BR is highly stable and its relatively high yield in the wild type organism, many scientists regard BR as a one of the best bio-light sensor material for further biotechnology application. The first part of this study focused on characterization of a protein from a gene bop found in Haloarcula marismortui (HmBRI). After sequence analysis to confirm its validity as BR or BR-like protein, the gene was cloned and constructed in E. coli system for over-expression and purification followed by absorbance spectrum measurements, photocycle, and proton pumping activity, this protein was confirmed as a proton pumping bacteriorhodopsin, and it was named HmBRI. The second part of this study mainly dedicated on protein yield test and a carefully evaluation of the potential in biotechnology usage. Various mutants were constructed and it was found that a D94N mutation leads to very high expression level in E.coli expression system and it is stable when treated with heat, light, and even in a almost water-free environment. A series of light intensity experiments unveil a plausible strategy to adopt HmBRI as a light intensity or roughly a UV meter. This study therefore confirms HmBRI as a new bacteriorhodopsin protein with light-driven proton pumping capability and it can be engineered for application in biotechnology.