蛋白質(zhì)的表達(dá)ppt課件-資料下載頁(yè)
2025-05-04 12:08本頁(yè)面
【正文】 相對(duì)簡(jiǎn)單，多不分支 。 ? 與其它系統(tǒng)相比，哺乳動(dòng)物細(xì)胞表達(dá)系統(tǒng)的優(yōu)勢(shì)在于能夠指導(dǎo)蛋白質(zhì)的正確折疊，提供復(fù)雜的 N型糖基化和準(zhǔn)確的 O型糖基化等多種翻譯后加工功能，因而表達(dá)產(chǎn)物在分子結(jié)構(gòu)、理化特性和生物學(xué)功能方面最接近于天然的高等生物蛋白質(zhì)分子。 ? 與大腸桿菌相比，哺乳動(dòng)物細(xì)胞的表達(dá)水平低、獲得高表達(dá)細(xì)胞株所需的時(shí)間長(zhǎng)、細(xì)胞大規(guī)模培養(yǎng)的成本高等導(dǎo)致哺乳動(dòng)物細(xì)胞生產(chǎn)蛋白質(zhì)的成本較高。 3 哺乳動(dòng)物細(xì)胞表達(dá)系統(tǒng) ?? ? 表達(dá)需要轉(zhuǎn)錄后修飾的基因 ? 研究蛋白的生物合成和體內(nèi)細(xì)胞間的轉(zhuǎn)運(yùn)機(jī)制 ? 表達(dá)大腸桿菌和酵母不能表達(dá)的含有內(nèi)含子的基因序列 ? 哺乳動(dòng)物細(xì)胞是最理想的表達(dá)人類(lèi)基因的系統(tǒng)，應(yīng)為首選。 哺乳動(dòng)物細(xì)胞表達(dá)系統(tǒng) 體外翻譯系統(tǒng) （ In vitro translation system）又稱(chēng)之為體外蛋白質(zhì)合成系統(tǒng) (in vitro protein synthesis system)，是一種細(xì)胞裂解物，這種系統(tǒng)只有翻譯功能，當(dāng)加入外源 mRNA，能量物質(zhì)和氨基酸，該系統(tǒng)就能合成蛋白質(zhì)，經(jīng) SDS— PAGE后可檢測(cè)出翻譯活性。 常用翻譯系統(tǒng)有以下幾種： 1. 兔網(wǎng)織細(xì)胞裂解物 (reticylocyte lysate system) 2. 小麥胚抽提物 (wheat germ extract) 3. 兩棲動(dòng)物卵母細(xì)胞 4. 原核體外翻譯系統(tǒng) Ⅳ 體外翻譯系統(tǒng) Ⅴ Commonly confronted questions ? Should the protein(s) be expressed in bacteria, in yeast, in insect cells or in human cells? ? Which expression vector should be used? ? If bacterial expression is used, which strain(s) should be chosen? ? Should one express the fulllength protein or a fragment thereof? ? Should the protein be tagged, and which affinity tag is the best? ? What is a good purification strategy, and what are the mon pitfalls? ? Unfortunately, because every protein is different, there can be no ?right‘ answer to any of these questions a priori. How to choose protein expression system ? Choice of host (system) ? Choice of vector ? Choice of tag ? Choice of strains 1. Choice of host ? bacteria, fungi, plants, insect or mammalian cells grown in culture, and transgenic animals or plants ? ―My Expression System is Better than Yours‖ SyndromeThere is no one expression system that does everything well, Each system has its strengths and weaknesses ? The amount and the degree of purity of the product, as well as its biological integrity and potential toxicity should be considered. ? The expression of each cDNA or gene presents its own peculiar set of problems. The synthesis of foreign protein is still largely empirical. 2. Choice of vector ? The choice of vector family is largely governed by the host. ? The expression of a rebinant protein fused to a tag of known size and biological function can greatly simplify subsequent purification and detection Expression of fusion proteins ? Advantages: 1. foreign proteins are often rapidly degraded by host protease, and this may sometimes be avoided by a gene fusion strategy. 2. General and efficient purification schemes maybe obtained which allow rapid recovery of gene products. 3. the proteins can be localised to different partments of the host cell (periplasm, cell wall, culture medium) through specific peptides fused to the protein. ? Disadvantages The fusion peptide may prevent the correct folding of the rebinant protein and alter its properties. For that reason the bacterial segment should be removable, usually chemically or enzymatically. Even after removal of the fusion peptide some proteins have been unable to achieve the correct folding 3. Choice of tag The two most monly used tags are (histidine)6 and GST 4. Choice of strains ? BL21 :lon, ompT ? BL21(DE3) : T7 polymerase ? Rosetta: optimal codons ? Origami : thioredoxin reductase (trxB) , glutathione reductase (gor) ? Rosettagami ? Origami B: (IPTG concentration dependent) 思考題 ? 原核表達(dá)載體主要有那些組件？ ? 重組蛋白質(zhì)的表達(dá)系統(tǒng)主要有那些？各自的優(yōu)缺點(diǎn)是什么？ 參考文獻(xiàn) ? 畢赤酵母表達(dá)操作手冊(cè) ? pET System Manual， ? 3. 蛋白質(zhì)工程，王大成，化學(xué)工業(yè)出版社，2022
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