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首页> 外文学位 >Identification of factors limiting heterologous lipase expression in the cytoplasm and the periplasm as well as display on cell surface of Escherichia coli.
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Identification of factors limiting heterologous lipase expression in the cytoplasm and the periplasm as well as display on cell surface of Escherichia coli.

机译：鉴定限制异源脂肪酶在细胞质和周质中表达以及在大肠杆菌细胞表面上展示的因素。

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摘要

Lipase B from Pseudozyma antarctica (PalB), had been expressed in several recombinant protein hosts and showed very good transesterification activity for biodiesel production. However, the functional expression could not be demonstrated until recently in the most popular recombinant protein expression system, e.g. Escherichia coli, and the expression performance stands improvement. This thesis focuses on the identification of factors limiting heterologous expression of PalB in E. coli through a systematic study by using several strategies, including the different expression compartments, fusion tags, folding factors, and host strains.;While PalB was stably expressed in the cytoplasm, most of the expressed gene product aggregated in cells as inactive inclusion bodies. In contrast, PalB was extremely unstable when expressed in the periplasm, also leading to poor expression performance. Such unstable PalB can be rescued by coexpression of several periplasmic folding factors, such as DegP, FkpA, DsbA, and DsbC, but not cytoplasmic chaperones. As a result, the performance for functional PalB expression in the periplasm was significantly improved. This is the first report demonstrating the use of folding factors to rescue the extremely unstable gene product that is otherwise completely degradable. On the other hand, functional expression of PalB in the periplasm was explored using four fusion tags, e.g., DsbC, DsbA, maltose binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags for functional expression of PalB, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of gene products, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegPS210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance for functional expression of PalB was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/liter.;Without extracting protein from cells the whole cell can be directly used as a platform for the immobilized enzyme. Proof-of-concept experimentation was conducted by PalB display on the E. coli cell surface. By fusing the palB gene in between the signal peptide phoA and an autotransporter Protein EstA's gene under the lac promoter, PalB was successfully displayed on the E. coli cell surface. However, cells encountered a severe physiological stress. Coexpression of various periplasmic folding factors, e.g., DegP, SurA, DsbA and DsbC could erease the physiological stress, but only DsbA was demonstrated to be effective to restore cell physiology and increase PalB expression level. (Abstract shortened by UMI.).;Functional expression of PalB in the cytoplasm of E. coli was explored using BL21(DE3) and its mutant derivative Origami B(DE3) as the host. Bioactive PalB was obtained in the reduced cytoplasm of BL21(DE3), implying that the formation of disulfide bond was not strictly required for functional expression. However, the expression was ineffective and was primarily limited by formation of PalB inclusion bodies and growth arrest, both of which were associated with PalB misfolding and deteriorated physiology. The culture performance in terms of cell growth and PalB expression level could be significantly improved by simultaneous coexpression of multiple chaperones of trigger factor and GroEL/ES, but not individual coexpression of either one of them. It was proposed that the two chaperones mediate the early stage and late stage of cytoplasmic PalB folding and would be required simultaneously for boosting both the overall PalB synthesis rate and the cytoplasmic folding efficiency. On the other hand, a much higher bioactive PalB was produced in Origami B(DE3) harboring the same PalB expression vector. Furthermore, the significant high bioactive PalB was produced by coexpression of periplasmic folding factor without a signal peptide (e.g., coexpression of DsbA, and DsbC). Coexpression of DsbA was found to be effective in enhancing PalB expression and such an improvement was more pronounced in Origami B(DE3), suggesting that both folding and disulfide bond formation could be the major factors limiting PalB expression. The fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity possibly via both enhanced solubility and correct disulfide bond formation. Our results suggest that solubilization of PalB fusions did not necessarily result in the development of PalB activity which could be closely associated with correct disulfide bond formation.
机译：来自南极假酶（PalB）的脂肪酶B已在几种重组蛋白宿主中表达，并且对生物柴油生产显示出非常好的酯交换活性。然而，直到最近，在最流行的重组蛋白表达系统中，例如在大肠杆菌中，才证明功能性表达。大肠埃希氏菌，表达性能得到改善。本论文着重通过系统的研究，通过多种策略，包括不同的表达区室，融合标签，折叠因子和宿主菌株，来鉴定限制PalB在大肠杆菌中异源表达的因素。在细胞质中，大多数表达的基因产物在细胞中聚集为无活性的包涵体。相反，当在周质中表达时，PalB非常不稳定，也导致较差的表达性能。这种不稳定的PalB可以通过共表达几种周质折叠因子（例如DegP，FkpA，DsbA和DsbC）而非细胞质分子伴侣来挽救。结果，周质中功能性PalB表达的性能显着提高。这是第一份证明使用折叠因子挽救原本可以完全降解的极不稳定基因产物的报告。另一方面，使用四个融合标签，例如DsbC，DsbA，麦芽糖结合蛋白（MBP）和FLAG，以增加表达效力的顺序探索了PalB在周质中的功能性表达。在这些用于PalB功能表达的融合标签中，就增强酶活性和增强基因产物的溶解度而言，FLAG和MBP似乎是最有效的。这些PalB融合蛋白的过表达通常会导致不溶性包涵体的同时形成。多种周质折叠因子的共表达，包括DegP（及其DegPS210A的突变变体），FkpA，DsbA，DsbC和SurA，FkpA，DsbA和DsbC的混合物，可以改善表达性能。 DsbA的共表达似乎在减少四种PalB融合物中包涵体的形成方面最有效，这意味着PalB的功能性表达可能受到二硫键的初步桥接的限制。通过过表达FLAG-PalB和DsbA共表达来优化PalB功能表达的培养性能，从而获得360 U /升的高体积PalB活性;无需从细胞中提取蛋白质，整个细胞就可以直接用作固定化平台酶。通过在大肠杆菌细胞表面上的PalB展示进行概念验证实验。通过将palB基因融合在lac启动子下的信号肽phoA和自转运蛋白EstA的基因之间，PalB成功地展示在大肠杆菌细胞表面。然而，细胞遇到了严重的生理压力。多种周质折叠因子例如DegP，SurA，DsbA和DsbC的共表达可以减轻生理压力，但是只有DsbA被证明对恢复细胞生理和增加PalB表达水平有效。 （UMI缩短。）；以BL21（DE3）及其突变衍生物Origami B（DE3）为宿主，探讨了PalB在大肠杆菌细胞质中的功能性表达。在BL21（DE3）的还原细胞质中获得了具有生物活性的PalB，这表明功能表达并非严格要求形成二硫键。但是，该表达是无效的，并且主要受到PalB包涵体的形成和生长停滞的限制，这两者均与PalB的错误折叠和生理现象有关。通过同时共表达多种触发因子和GroEL / ES分子伴侣，但不能同时共表达它们中的任何一种，可以显着提高细胞生长和PalB表达水平方面的培养性能。有人提出，这两个分子伴侣介导细胞质PalB折叠的早期和晚期，同时需要同时用于提高总体PalB合成速率和细胞质折叠效率。另一方面，在具有相同PalB表达载体的Origami B（DE3）中产生了更高的生物活性PalB。此外，显着的高生物活性PalB是通过周质折叠因子的共表达而没有信号肽产生的（例如，DsbA和DsbC的共表达）。发现DsbA的共表达可有效增强PalB的表达，而这种改善在Origami B（DE3）中更为明显，这表明折叠和二硫键的形成可能是限制PalB表达的主要因素。通过构建几种PalB融合蛋白以评估其表达性能，还探索了融合标签技术。虽然大多数PalB融合的溶解度有所提高，只有DsbA标签可能有效地通过增强溶解度和正确的二硫键形成来增强PalB活性。我们的结果表明，PalB融合蛋白的溶解并不一定会导致PalB活性的发展，而这可能与正确的二硫键形成密切相关。

著录项

  • 作者

    Xu, Yali.;

  • 作者单位

    University of Waterloo (Canada).;

  • 授予单位 University of Waterloo (Canada).;
  • 学科 Engineering Chemical.;Biology Cell.
  • 学位 Ph.D.
  • 年度 2008
  • 页码 226 p.
  • 总页数 226
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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