綠色熒光蛋白的發(fā)光原理：

綠色熒光蛋白的GFP(Green fluorescent protein)的第65至67位的三個(gè)氨基酸（絲氨酸-酪氨酸-甘氨酸）殘基，可自發(fā)地形成一種熒光發(fā)色團(tuán)。當(dāng)?shù)鞍踪|(zhì)鏈折疊時(shí)，這段被深埋在蛋白質(zhì)內(nèi)部的氨基酸片段，得以“親密接觸”，導(dǎo)致經(jīng)環(huán)化形成咪唑酮，并發(fā)生脫水反應(yīng)。在分子氧存在的條件下，發(fā)色團(tuán)可進(jìn)一步發(fā)生氧化脫氫，最終成熟，形成可發(fā)射熒光的形式。具體過程為：在 O2存在下，GFP分子內(nèi)第67位甘氨酸的酰胺對(duì)第65位絲氨酸的羧基進(jìn)行親核攻擊，形成第5位碳原子咪唑基；第66位酪氨酸的α2β鍵脫氫反應(yīng)之后，導(dǎo)致芳香團(tuán)與咪唑基結(jié)合，并最終自發(fā)催化形成對(duì)羥基苯甲酸咪唑環(huán)酮生色。

GFP需要在氧化狀態(tài)下產(chǎn)生熒光，強(qiáng)還原劑能使GFP轉(zhuǎn)變?yōu)榉菬晒庑问?，但一旦重新暴露在空氣或氧氣中，GFP熒光便立即得到恢復(fù)。一般來說弱還原劑并不會(huì)影響GFP熒光，中度氧化劑如生物材料的固定，脫水劑戊二酸或甲醛等對(duì)GFP熒光影響也不大。

綠色熒光蛋白的發(fā)光特性：

綠色熒光蛋白的GFP吸收的光譜更大峰值為395nm（紫外），并有一個(gè)峰值為470nm的副吸收峰（藍(lán)光）；發(fā)射光譜更大峰值為509nm（綠光），并帶有峰值為540nm的側(cè)峰（Shouder）。雖然450～490nm只是GFP的副吸收峰，但由于該激發(fā)光對(duì)細(xì)胞的傷害更小，因此通常多使用該波段光源（多為488nm）。此外，GFP的光譜特性與熒光素異硫氰酸鹽（FITC）很相似，兩者通常共有一套濾光片。GFP熒光極其穩(wěn)定，在激發(fā)光照射下，GFP抗光漂白（Photobleaching）能力比熒光素強(qiáng)，特別是在450～490nm藍(lán)光波長下更穩(wěn)定。類似的，GFP融合蛋白的熒光靈敏度遠(yuǎn)比熒光素標(biāo)記的熒光抗體高，抗光漂白能力強(qiáng)，因此更適用于定量測定與分析。由于GFP熒光的產(chǎn)生不需要任何外源反應(yīng)底物，因此GFP作為一種廣泛應(yīng)用的活體報(bào)告蛋白，其作用是任何其它酶類報(bào)告蛋白無法比擬的。但因?yàn)镚FP不是酶，熒光信號(hào)沒有酶學(xué)放大效果，因此GFP靈敏度可能低于某些酶類報(bào)告蛋白，比如螢火蟲熒光素酶等。

綠色熒光蛋白的應(yīng)用：

廣泛應(yīng)用于分子標(biāo)記，體內(nèi)示蹤，信號(hào)轉(zhuǎn)導(dǎo)，藥物篩選等生物科研的各個(gè)方面。

諾貝爾獎(jiǎng)：

由于GFP的廣泛應(yīng)用以及在生命科學(xué)科研中發(fā)揮的巨大作用，2008年諾貝爾化學(xué)獎(jiǎng)授予了，發(fā)現(xiàn)并發(fā)展了綠色熒光蛋白（GFP）的三位科學(xué)家，分別是日本科學(xué)家Osamu Shimomura（下村修）、美國哥倫比亞大學(xué)的Marin Chalfie以及加州大學(xué)圣地亞哥分校的Roger Y.Tsien（錢永健，錢學(xué)森堂侄）。

綠色熒光蛋白的的檢測

析浦科學(xué)儀器有限公司生產(chǎn)有便攜式熒光蛋白檢測手電筒，無需熒光顯微鏡，通過熒光手電筒照射轉(zhuǎn)基因植物就可以憑肉眼檢測觀察綠色熒光蛋白有沒有表達(dá)，簡單方便快捷觀察熒光蛋白的表達(dá)。產(chǎn)品詳細(xì)介紹請(qǐng)瀏覽：手持式GFP熒光手電筒GFPfinder-2101

The very aptly named green fluorescent protein — or GFP as it is almost universally known — is a barrel-shaped protein made up of 238 amino acids. Threaded through the long axis of the β-sheet barrel is an α-helix that contains a chromophore that is responsible for the emission of green light when GFP is exposed to either blue or ultaviolet light. This particular property, coupled with the fact that GFP is well tolerated by many different organisms, has led to its use as a fluorescent tag for monitoring biological processes at the cellular level.

The story of GFP begins in the oceans with the jellyfish Aequorea victoria, which has the unusual property that its outer edges glow green when it is agitated. In the early 1960s, Osamu Shimomura collected raw material from thousands of these jellyfish and extracted a small amount of a blue luminescent protein, which was subsequently named aequorin. During this process he also found another substance that glowed green when exposed to ultraviolet light — this was the protein that later became known as GFP. Shimomura and colleagues went on to show that the green glow produced by the jellyfish arises from an energy-transfer process in which the aequorin donor excites the GFP acceptor, which then emits green light.

In the early 1990s, Martin Chalfie and co-workers demonstrated that GFP could be expressed in organisms other than Aequorea victoria — such as Escherichia coli and Caenorhabditis elegans — and this was the breakthrough that paved the way for the practical implementation of GFP as a fluorescent tag for studying biological processes. It was generally thought that a number of steps requiring other proteins would be needed to produce the chromophore in GFP, but these experiments proved this to be wrong. Significantly, this result meant that GFP could be used as a universal tag, because no other auxiliary agents were needed to induce fluorescence. By engineering the genetic machinery of C. elegans so that it would produce GFP when a protein with a specific activity was expressed inside a cell, Chalfie was able to see cellular processes in a whole new light — albeit a green one!

The further development of GFP was based on a greater understanding of the molecular structure of the protein and specifically the chromophore responsible for its colourful name. Roger Tsien and co-workers explained how three amino acids in the peptide backbone of GFP — namely serine, tyrosine and glycine in positions 65, 66 and 67, respectively — react in the presence of oxygen to form the fluorescent chromophore p-hydroxybenzylideneimidazolinone. With this more detailed description of GFP, Tsien went on to develop other GFP derivatives with different spectral characteristics and increased stability. Not only could the brightness of the fluorescence be enhanced, but also the colour of emission could be tuned. Today all the colours of the rainbow can be found in a range of GFP and GFP-like proteins.

Apart from the obvious biomedical implications, GFP sensors have also been developed that can detect chemical species such as metals ions and small molecules. So not only has GFP enabled scientists to see biological processes in a whole new light, but many other chemical opportunities await. The future for GFP is a bright one.