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lanlan_zhu木蟲 (著名寫手)
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DISCUSSION A. niger is used commercially in the production of a wide range of secreted enzymes and is being developed as a host for the secretion of heterologous enzymes (Archer & Peberdy, 1997; Gouka et al., 1997a). Despite the biotechnological importance of protein secretion in filamentous fungi, details of the secretory pathway are largely unknown. Much of our current knowledge regarding protein secretion has come from the study of temperature-sensitive secretion mutants in the yeast Saccharomyces cerevisiae. Identification of several homologues in other eukaryotes, including A. niger (Veldhuisen et al., 1997), indicates that the process of protein secretion is highly conserved. It is a reasonable assumption therefore, that protein secretion in yeasts and filamentous fungi will share many features. However, the biological and orphological differences between these two groups suggest that additional proteins may be involved in the process of protein secretion in the fungal mycelium. This has been highlighted recently by the identification of an annexin homologue in Neurospora crassa which shares homology to the annexin gene family in higher eukaryotes but appears to be absent in S. cerevisiae (Braun et al., 1998). In a series of gene fusions we replaced the starch-binding domain of glucoamylase with sGFP to create fluorescent markers for the study of the secretion process in A. niger. Three glucoamylase sGFP fusions were made. Two of them were designed for secretion of the fusion protein and employed two different lengths of the glucoamylase protein (GLA499 and GLA514), although both constructs lacked the starch-binding domain. This approach has been used successfully to secrete heterologous proteins in both cases. The third construct included a C-terminal HDEL motif designed to retain the fusion protein within the lumen of the ER. The different lengths of glucoamylase in the secretory fusions were found to have no significant effect as both GLA499: :sGFP and GLA514: : sGFP produced the same patterns of fluorescence in A. niger, and were distinct from that of the ER-retained GLA514: : sGFP-HDELand the cytoplasmic-sGFP-containing strains. In young mycelia expressing the GLA: : sGFP fusion protein, bright fluorescence was observed in the hyphal walls indicating that the fusion protein was secreted, but retained within the cell wall. The presence of the fusion protein in the cell wall was confirmed by immuno-gold labelling. Retention of extracellular proteins in the hyphal wall has been reported previously for glucose oxidase in A. niger (Witteveen et al., 1992) and invertase in N. crassa (Trevithick & Metezenberg, 1966) and for a variety of secreted proteins in S. cerevisiae (de Nobel & Barnett, 1991). Using sGFP, we were able to directly observe in vivo the presence of the fusion protein within the cell wall, which would have been difficult based solely on Western analysis. We developed a simple extraction method that released the fusion protein from the cell wall. It is likely that in previous studies in which glucoamylase±gene fusions have been used for heterologous protein secretion, cell wall localization was not recognized because the extraction method used did not discriminate between cell-wall-bound and intracellular proteins (Ward et al., 1990; Broekhuijsen et al., 1993; Archer et al., 1994; Gouka et al., 1997b).The more intense fluorescence observed at hyphal apices supports the hypothesis that secretion of the fusion protein takes place at the hyphal tips (Wo$ sten et al., 1991). However, as subapical regions of the cell wall were also fluorescent it appears that at least some of the GLA: : sGFP fusion protein is retained in the hyphal wall following secretion at the hyphal apex. Unexpectedly, septa were also brightly fluorescent, indicating the presence of GLA: : sGFP fusion protein. Since the formation of septa takes place independently of apical growth, the question arises of how septa become fluorescent. One explanation might be that the GLA: : sGFP fusion protein in the cell wall is trapped but freely diffusible within the extracellular matrix. Alternatively, the GLA : : sGFP fusion protein might be secreted during the formation of the septum. It is also possible that not all the secretion of the fusion protein is correlated with cell growth and that secretion also occurs in subapical cells without cell wall expansion. The effect of extracellular pH on fluorescence of the GLA: :sGFP-expressing strain compared to the GLA: : sGFP-HDEL strain also provides further evidence for the extracellular localization of GLA: : sGFP in the hyphal wall. Fluorescence of GFP has been shown to be sensitive to low pH, and below pH 5.0 loss of fluorescence may be irreversible (Kneen et al., 1998). The extracellular pH values of media of shake-flask cultures of A. niger have been shown to decrease to as low as 2.0 during growth (Archer et al., 1990). Low pH induces the production of proteases that are known to affect yields of heterologous proteins (Archer & Peberdy, 1997; Gouka et al., 1997a; van den Homberg et al., 1997). Extracellular proteases probably account for the degradation of the GLA: : sGFP fusion proteins in the culture supernatant, even in soya milk medium where degradation of sGFP was found to occur at a slower rate than in defined medium. Cleavage of the GLA: :sGFP fusion protein appeared to occur initially within the linker region between the glucoamylase and sGFP as cleaved sGFP with an apparent molecular mass of 27 kDa (the expected molecular mass for intact sGFP) was detected by Western analysis after 4 d growth in soya milk medium. Cleavage of the glucoamylase fusion protein at or near to the fusion junction has been reported for other heterologous proteins, even in the absence of a recognized processing site (Roberts et al., 1992). Further degradation of sGFP in the supernatant was indicated by the loss of detectable amounts of sGFP after 6 d. The D15 mutant, which has a reduced ability to acidify the medium, was able to sustain extracellular wall fluorescence for longer than AB4.1. The data suggest that the GLA: : sGFP fusion constructs can be used to monitor protein secretion in fermenters as long as the pH is held above pH 6±0. It may also be possible to use the GLA: : sGFP-expressing strain to screen for additional protease-deficient mutants. Taken together, the results indicate that in young mycelia the GLA: :sGFP fusion protein is primarily secreted at hyphal tips but partly retained within the cell wall, resulting in wall fluorescence. In older mycelia, extracellular wall fluorescence is lost as a result of the acidification of the culture medium and proteolytic degradation, possibly by acidinduced proteases. Fig. 6. Secretion occurs at the hyphal tips. Young germlings of A. niger AB4.1 G514: :sGFP grown for (a) 20 h and (b) 28 h showing apical localization and loss of subapical wall fluorescence. Bar, 20 μm. Fig. 7. (a) Growth of untransformed A. niger AB4.1 (+) and G499: :sGFP (E) on soya milk medium containing 1% (w/v) maltodextrin for 10 d, and the relative fluorescence of extracellular culture supernatant samples from AB4.1 (U) and G499: :sGFP (o). (b) Western blot analysis (i) using anti-sGFP antibodies of extracellular culture supernatant samples from A. niger AB4.1 G499: :sGFP grown on soya milk medium for 4, 6, 8 and 10 d (lanes 1, 2, 3 and 4, respectively) and showing the GLA : :sGFP fusion protein and (ii) using anti-glucoamylase (left panel) and anti-sGFP (right panel) antibodies on samples of culture filtrate from A. niger G499: :sGFP (lane 1) and AB4.1 (lane 2) after 4 d growth on soya milk medium, demonstrating that the protein band with the highest molecular mass is the GLA: :sGFP fusion protein. 我晚上就要，希望大家多多幫忙。我實在沒時間去翻譯。謝謝了。 [ Last edited by lanlan_zhu on 2010-5-31 at 11:49 ] |
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wypward(金幣+2):歡迎新蟲，謝謝參與 2010-05-31 16:26:41
lanlan_zhu(金幣+1, 翻譯EPI+1): 2010-06-01 19:47:18
lanlan_zhu(金幣+99): 2010-06-01 19:47:23
wypward(金幣+2):歡迎新蟲，謝謝參與 2010-05-31 16:26:41
lanlan_zhu(金幣+1, 翻譯EPI+1): 2010-06-01 19:47:18
lanlan_zhu(金幣+99): 2010-06-01 19:47:23
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由于翻譯的比較匆忙 會有一些有些出入的地方不太正確，請見諒 ，可供參考。 討論 黑曲霉，是用于商業(yè)，是一個生產(chǎn)各種酶的分泌，目前正在作為外源酶的分泌主要開發(fā) (Archer & Peberdy, 1997; Gouka et al., 1997a)。 盡管在絲狀真菌分泌蛋白生物技術(shù)有重要性，分泌途徑細節(jié)在很大程度上是未知。我們目前的知識對于蛋白質(zhì)的分泌有很多來自于對溫度敏感突變體分泌研究在釀酒的酵母中。鑒定同源其他幾個真核生物，包括黑曲霉（Veldhuisen et al.1997） 表明該蛋白質(zhì)的分泌過程是高度保守的。這是一個合理的假設(shè)，因此，在酵母蛋白分泌和絲狀真菌將有許多特點。然而，這兩個群體之間的生物和orphological差異表明，額外的蛋白質(zhì)可能在蛋白質(zhì)的分泌過程中涉及的真菌菌絲。這是突出強調(diào)了在粗糙脈孢菌膜聯(lián)蛋白同源基因同源性的股票的膜聯(lián)蛋白基因家族在高等真核生物，但似乎是在S缺席酵母（布朗等人最近鑒定。，1998）。 在我們的基因融合系列取代了淀粉糖化酶與sGFP結(jié)合域創(chuàng)造了在黑曲霉分泌過程的研究熒光標記。 其中兩位是專為融合蛋白的分泌，并雇用兩名糖化酶蛋白（GLA499和GLA514不同長度），雖然兩者結(jié)構(gòu)缺乏淀粉結(jié)合域。這種方法已成功地用于在兩種情況下分泌外源蛋白。第三個構(gòu)造包括一個C -末端HDEL主題，旨在保留范圍內(nèi)的內(nèi)質(zhì)網(wǎng)腔的融合蛋白。糖化酶中分泌融合不同長度被發(fā)現(xiàn)有兩個GLA499沒有顯著的效果：GLA514：：sGFP在黑曲霉產(chǎn)生熒光相同的模式，并從這一獨特的ER -保留GLA514：在青年表示GLA的菌絲：：sGFP融合蛋白，明亮的熒光觀察顯示，該融合蛋白分泌的菌絲壁，但在細胞壁保留。在細胞壁中的融合蛋白的存在證實了免疫金標記。胞外蛋白保留在菌絲壁已被報告過，葡萄糖氧化酶的黑曲霉（維特芬等。，1992年）和北路粗糙脈孢菌（特里維西克＆Metezenberg，1966年蔗糖）和一對釀酒酵母（德諾貝爾＆巴尼特，1991）分泌多種蛋白質(zhì)。使用sGFP，我們能夠直接觀察活體細胞內(nèi)的墻壁，融合蛋白的存在，將有難以完全根據(jù)西方分析。我們開發(fā)一個簡單的提取方法，釋放出細胞壁的融合蛋白。很可能在其中糖化酶±基因融合以往的研究已經(jīng)用異源蛋白的分泌，細胞壁的定位是不承認，因為沒有用萃取法之間的細胞壁和細胞內(nèi)蛋白質(zhì)的約束歧視(Archer & Peberdy, 1997; Gouka et al., 1997a; van den Homberg et al., 1997). 。胞外蛋白酶可能為GLA的退化：在培養(yǎng)上清sGFP融合蛋白，即使在豆?jié){中的地方被發(fā)現(xiàn)的sGFP退化發(fā)生在比中定義的速度較慢卵裂的GLA的：：sGFP融合蛋白似乎發(fā)生在糖化酶之間的和作為一個明顯的切割分子量27 kDa的連接器sGFP sGFP地區(qū)最初（預計分子質(zhì)量為完整sGFP）西方分析檢測后4 d的豆?jié){中生長。對達到或接近糖化酶融合蛋白裂解交界處的融合已經(jīng)公布了其他外源蛋白，即使在一個公認的加工點（羅伯茨等人的情況下，。，1992）進一步退化的sGFP上清液中曾表示，檢出的sGFP損失金額四后六在D15中突變體，它有一個酸化能力下降的介質(zhì)，能維持超過AB4.1胞壁熒光。這些數(shù)據(jù)表明，亞麻酸：：sGFP融合結(jié)構(gòu)，可用于監(jiān)測發(fā)酵蛋白分泌，只要持有以上，pH值pH值6 ± 0。它也可以使用GLA的：：sGFP表現(xiàn)的菌株篩選額外蛋白酶缺失突變體。兩者合計，結(jié)果表明，在年輕的GLA的菌絲sGFP融合蛋白主要是在菌絲分泌的提示，但部分在細胞壁保留在墻壁造成熒光在較舊的菌絲體，胞壁熒光丟失作為培養(yǎng)液和蛋白水解酸化降解的結(jié)果，可能由acidinduced蛋白酶。圖。 6。分泌發(fā)生在菌絲提示。楊萱藻幼苗對黑曲霉AB4.1 G514：：sGFP成長為（1）20 h和（二）28 Ĥ顯示根尖定位和根尖下壁熒光損失。酒吧，20微米。圖。 7。 （一未轉(zhuǎn)基因黑曲霉）增長AB4.1（+）和G499：：sGFP（E）對豆?jié){中含有1％（瓦特/ v）為10 d和文化的相對熒光細胞外液從樣品麥芽糊精AB4.1（U）和G499：：sGFP（海外）。 （二）免疫印跡分析（一）使用外文化反sGFP抗體從黑曲霉上清樣品AB4.1 G499：sGFP生長在豆?jié){中4，6，8和10 d（通道1，2，3和4分別）和顯示sGFP融合蛋白及（ii）利用反糖化酶（左側(cè)面板）和文化樣本反sGFP（右圖）抗體濾液從黑曲霉G499：sGFP（線1）和AB4.1（后2巷4 D對豆?jié){中增長），表明具有最高分子量蛋白條帶，是亞麻酸：：sGFP融合蛋白。 |
2樓2010-05-31 14:40:07
lanlan_zhu
木蟲 (著名寫手)
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3樓2010-05-31 16:16:50
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