Characterization of Escherichia coli expressing an Lpp'OmpA(46-159)-PhoA fusion protein localized in the outer membrane
Characterization of Escherichia coli expressing an Lpp'OmpA(46-159)-PhoA fusion protein localized in the outer membrane
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DOI：
10.1007/s002530050657
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发表时间：
1996-03-01
影响因子：
5
通讯作者：
Earhart, CF
中科院分区：
文献类型：
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作者：
Stathopoulos, C;Georgiou, G;Earhart, CF
LPP‘OmpA(46-159)杂合蛋白可以作为一种有效的靶向载体将各种原核和真核可溶蛋白定位到大肠杆菌表面，从而为几种可能的生物技术应用提供了一个系统。在这里，我们证明了LPP‘OmpA(46-159)和细菌碱性磷酸酶(PhoA)之间的融合也针对外膜。然而，蛋白酶可及性实验和免疫电子显微镜显示，与其他周质蛋白不同，在外膜完整的细胞中，这些融合的PhoA结构域不暴露在细胞表面。影响周质中二硫键的形成和PhoA结构域的折叠的条件不仅不利于靶向细胞表面，而且当融合从高拷贝数的质粒中表达时，会导致致死性。此外，表达LPP‘OmpA(46-159)-PhoA融合蛋白的大肠杆菌在外膜通透性方面表现出依赖于应变和温度的变化。我们的结果与以前用其他载体进行的研究一致，表明当与细胞表面表达载体融合时，PhoA不会显示在表面。据推测，这种酶很快就呈现出一种紧密折叠的二聚体构象，这种构象不能通过外膜运输。PhoA的大小和四级结构可能定义了LPP‘OmpA(46-159)融合系统在细胞表面显示周质蛋白的局限性。碱性磷酸酶是五种周质蛋白(β-内酰胺酶、碱性磷酸酶、Cex纤维素酶、Cex纤维素结合域和单链抗体片段)中唯一的一种蛋白，迄今已被测试为LPP‘OmpA(46-159)表达系统的载体，因为它是唯一未在LPP’OmpA(46-159)表面展示的蛋白。
The Lpp'OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E. coli surface, thus providing a system for several possible biotechnology applications. Here we show that fusions between Lpp'OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane. However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane. Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid. Furthermore, E. coli expressing the Lpp'OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability. Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors. Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane. The large size and quaternary structure of PhoA may define a limitation of the Lpp'OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface. alkaline phosphatase is a unique protein among a group of five periplasmic proteins (beta-lactamase, alkaline phosphatase, Cex cellulase, Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp'OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.