【作者】 连佳长；

【导师】 徐志南；

【作者基本信息】 浙江大学 ， 生物化工， 2010， 硕士

【摘要】 大肠杆菌水通道蛋白Z(AqpZ)水选择专一性强、渗透性高、结构稳定,所以AqpZ在仿生废水回收以及海水淡化等领域具有潜在的重要应用价值。但是由于AqpZ强烈疏水性会导致细胞毒性,使得在传统大肠杆菌体系表达量仅仅只有2.5 mg/l,很难满足制备基于水通道蛋白的水过滤装置的需求。本工作的目的是运用融合表达策略和无细胞体系表达策略,高效制备功能性AqpZ,为制备新型水过滤装置奠定坚实的基础,并发展一个具有普遍意义的膜蛋白高效表达技术。首先采用融合表达策略,即在目标膜蛋白的上游引入亲水性分子伴侣,构建带有不同融合标签的体内表达载体。发现pMAL-P2-AqpZ对宿主细胞生长的影响最小,融合蛋白的表达水平最高,进一步优化诱导条件(包括诱导时间、培养温度、IPTG浓度、诱导后培养时间),表达量可高达200 mg/l。由于AqpZ疏水性会导致细胞毒性,随后采用没有生长要求的无细胞蛋白质合成体系。构建的一系列带有不同N端序列的无细胞体系表达载体中,pIVEX2.4c-AqpZ为最佳表达载体。由于无细胞体系缺少膜蛋白正确折叠所需要的疏水环境,AqpZ都是以沉淀的形式表达。首先采用去污剂重悬的模式来制备可溶性AqpZ,然而采用的10余种去污剂均不能有效溶解AqpZ沉淀。考虑到无细胞体系的开放性特点,采用直接添加去污剂的模式,发现Brij78为最优去污剂,在112xCMC最佳工作浓度下,可溶表达水平约为530 mg/l。此外,还研究了直接添加脂质体的模式,在实验最高脂质体浓度(5 mg/ml)下,整合到磷脂膜的AqpZ表达水平达到470 mg/l。在无细胞体系表达AqpZ时发现,mRNA翻译起始区形成二级结构会抑制翻译的有效启动,使得不带有N端标签的AqpZ表达量较低,所以开展了无细胞体系高效表达AqpZ新策略方面的研究。首先采用双顺反子策略,虽然构建的双顺反子质粒能够有效提高AqpZ在大肠杆菌体系的表达水平,但这些质粒并不能在无细胞体系实现高效表达。随后采用信号肽策略,构建带有不同信号肽序列的AqpZ体外表达载体,目标蛋白在无细胞体系的表达量提高了5倍以上;且通过添加去污剂或者脂质体可以激活无细胞抽提物中的信号肽酶活性,实现信号肽序列的原位切除,说明信号肽序列可以作为促进蛋白翻译的序列元件而发展一个具有普遍意义的、与目的蛋白DNA序列无关的膜蛋白高效表达新策略。更多还原

【Abstract】 Aquaporin Z (AqpZ), the water channel protein from Escherichia coli, is an orthodox aquaporin with high water selectivity and high permeability, making AqpZ-based water filter a promising tool for water reuse and seawater desalination. However, the development of this novel bio-filter is hampered by the availability of sufficient amount of water channel proteins since the overexpression of integral membrane proteins may exhibit toxicity towards the host due to the highly hydrophobic nature.The aim of this work is to synthesize functional AqpZ at high levels either by fusion expression strategy or cell-free expression system, which lays a solid foundation to construct the novel aquaporin-based water filter, and also promises a universal platform for efficient production of membrane proteins.Firstly, fusion expression was chosen to improve the expression level of AqpZ. By fusion with several hydrophilic partners, the yield of AqpZ was significantly improved, and MBP was determined to be the most efficient fusion partner to increase the expression level. After systematically investigating the effects of different induction conditions, such as temperature, induction-timing, IPTG concentration and post-induction duration, high productivity of MBP-AqpZ (200 mg/l) was achieved.Because the overexpression of membrane proteins would lead to cell toxicity, no growth-required cell-free protein synthesis (CFPS) system was employed as an alternative to synthesize AqpZ. Several in vitro expression vectors with different N-terminal sequence were constructed, and pIVEX2.4c-AqpZ was determined to be the most efficient vector in cell-free system. However, due to the lack of hydrophobic environment essential for membrane protein folding, AqpZ was aggregated as precipitates in a standard CFPS. In order to produce soluble AqpZ, detergent resolubilization was investigated first. However, all the tested detergents in this work failed to resolubilize the AqpZ precipitates efficiently. Therefore, detergents were supplemented directly instead, taking advantage of the open nature of CFPS. By systematically screening and optimizing of detergent structure and concentration, the highest soluble expression (530 mg/1) was achieved by Brij78 at a final concentration of 112xCMC. In addition, DOPC liposome supplementation was also investigated, and 470 mg/1 membrane-associated AqpZ was achieved at the highest DOPC concentration tested (5 mg/ml).Furthermore, the formation of mRNA secondary structure was found to inhibit translation initiation, which limited the expression level of native AqpZ in cell-free system. Therefore, research work on two-cistronic plasmid strategy and leader peptide strategy were undertaken. Although the constructed two-cistronic plasmids could improve the expression level of AqpZ in vivo, these plasmids didn’t work well in cell-free system. On the contrary, by fusion with many naturally occurring leader peptides, the expression level of AqpZ was enhanced by as much as 5 times through avoiding the formation of secondary structure. In addition, the leader peptide could be cleaved in situ in cell-free system supplemented with detergent or liposome, through the activation of leader peptidase. These results indicated that leader peptide sequence could be used as a downstream box to stimulate recombinant protein synthesis in cell-free system, promising a universal platform for efficient production of membrane proteins, regardless of the nature of target DNA sequence.更多还原