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lanlan_zhu木虫 (著名写手)
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求翻译生物类文献 有效期至2010年5月31日 20:00
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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，希望大家多多帮忙。我实在没时间去翻译。谢谢了。 [ Last edited by lanlan_zhu on 2010-5-31 at 11:49 ] |
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wypward(金币+2):欢迎新虫，谢谢参与 2010-05-31 16:26:41
lanlan_zhu(金币+1, 翻译EPI+1): 2010-06-01 19:47:18
lanlan_zhu(金币+99): 2010-06-01 19:47:23
wypward(金币+2):欢迎新虫，谢谢参与 2010-05-31 16:26:41
lanlan_zhu(金币+1, 翻译EPI+1): 2010-06-01 19:47:18
lanlan_zhu(金币+99): 2010-06-01 19:47:23
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由于翻译的比较匆忙 会有一些有些出入的地方不太正确，请见谅 ，可供参考。 讨论 黑曲霉，是用于商业，是一个生产各种酶的分泌，目前正在作为外源酶的分泌主要开发 (Archer & Peberdy, 1997; Gouka et al., 1997a)。 尽管在丝状真菌分泌蛋白生物技术有重要性，分泌途径细节在很大程度上是未知。我们目前的知识对于蛋白质的分泌有很多来自于对温度敏感突变体分泌研究在酿酒的酵母中。鉴定同源其他几个真核生物，包括黑曲霉（Veldhuisen et al.1997） 表明该蛋白质的分泌过程是高度保守的。这是一个合理的假设，因此，在酵母蛋白分泌和丝状真菌将有许多特点。然而，这两个群体之间的生物和orphological差异表明，额外的蛋白质可能在蛋白质的分泌过程中涉及的真菌菌丝。这是突出强调了在粗糙脉孢菌膜联蛋白同源基因同源性的股票的膜联蛋白基因家族在高等真核生物，但似乎是在S缺席酵母（布朗等人最近鉴定。，1998）。 在我们的基因融合系列取代了淀粉糖化酶与sGFP结合域创造了在黑曲霉分泌过程的研究荧光标记。 其中两位是专为融合蛋白的分泌，并雇用两名糖化酶蛋白（GLA499和GLA514不同长度），虽然两者结构缺乏淀粉结合域。这种方法已成功地用于在两种情况下分泌外源蛋白。第三个构造包括一个C -末端HDEL主题，旨在保留范围内的内质网腔的融合蛋白。糖化酶中分泌融合不同长度被发现有两个GLA499没有显着的效果：GLA514：：sGFP在黑曲霉产生荧光相同的模式，并从这一独特的ER -保留GLA514：在青年表示GLA的菌丝：：sGFP融合蛋白，明亮的荧光观察显示，该融合蛋白分泌的菌丝壁，但在细胞壁保留。在细胞壁中的融合蛋白的存在证实了免疫金标记。胞外蛋白保留在菌丝壁已被报告过，葡萄糖氧化酶的黑曲霉（维特芬等。，1992年）和北路粗糙脉孢菌（特里维西克＆Metezenberg，1966年蔗糖）和一对酿酒酵母（德诺贝尔＆巴尼特，1991）分泌多种蛋白质。使用sGFP，我们能够直接观察活体细胞内的墙壁，融合蛋白的存在，将有难以完全根据西方分析。我们开发一个简单的提取方法，释放出细胞壁的融合蛋白。很可能在其中糖化酶±基因融合以往的研究已经用异源蛋白的分泌，细胞壁的定位是不承认，因为没有用萃取法之间的细胞壁和细胞内蛋白质的约束歧视(Archer & Peberdy, 1997; Gouka et al., 1997a; van den Homberg et al., 1997). 。胞外蛋白酶可能为GLA的退化：在培养上清sGFP融合蛋白，即使在豆浆中的地方被发现的sGFP退化发生在比中定义的速度较慢卵裂的GLA的：：sGFP融合蛋白似乎发生在糖化酶之间的和作为一个明显的切割分子量27 kDa的连接器sGFP sGFP地区最初（预计分子质量为完整sGFP）西方分析检测后4 d的豆浆中生长。对达到或接近糖化酶融合蛋白裂解交界处的融合已经公布了其他外源蛋白，即使在一个公认的加工点（罗伯茨等人的情况下，。，1992）进一步退化的sGFP上清液中曾表示，检出的sGFP损失金额四后六在D15中突变体，它有一个酸化能力下降的介质，能维持超过AB4.1胞壁荧光。这些数据表明，亚麻酸：：sGFP融合结构，可用于监测发酵蛋白分泌，只要持有以上，pH值pH值6 ± 0。它也可以使用GLA的：：sGFP表现的菌株筛选额外蛋白酶缺失突变体。两者合计，结果表明，在年轻的GLA的菌丝sGFP融合蛋白主要是在菌丝分泌的提示，但部分在细胞壁保留在墙壁造成荧光在较旧的菌丝体，胞壁荧光丢失作为培养液和蛋白水解酸化降解的结果，可能由acidinduced蛋白酶。图。 6。分泌发生在菌丝提示。杨萱藻幼苗对黑曲霉AB4.1 G514：：sGFP成长为（1）20 h和（二）28 Ĥ显示根尖定位和根尖下壁荧光损失。酒吧，20微米。图。 7。 （一未转基因黑曲霉）增长AB4.1（+）和G499：：sGFP（E）对豆浆中含有1％（瓦特/ v）为10 d和文化的相对荧光细胞外液从样品麦芽糊精AB4.1（U）和G499：：sGFP（海外）。 （二）免疫印迹分析（一）使用外文化反sGFP抗体从黑曲霉上清样品AB4.1 G499：sGFP生长在豆浆中4，6，8和10 d（通道1，2，3和4分别）和显示sGFP融合蛋白及（ii）利用反糖化酶（左侧面板）和文化样本反sGFP（右图）抗体滤液从黑曲霉G499：sGFP（线1）和AB4.1（后2巷4 D对豆浆中增长），表明具有最高分子量蛋白条带，是亚麻酸：：sGFP融合蛋白。 |
2楼2010-05-31 14:40:07
lanlan_zhu
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3楼2010-05-31 16:16:50
