Display of beta-lactamase on the Escherichia coli surface: Outer membrane phenotypes conferred by Lpp'-OmpA'-beta-lactamase fusions
Display of beta-lactamase on the Escherichia coli surface: Outer membrane phenotypes conferred by Lpp'-OmpA'-beta-lactamase fusions
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DOI：
10.1093/protein/9.2.239
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发表时间：
1996-02-01
期刊：
影响因子：
--
通讯作者：
Earhart, CF
中科院分区：
文献类型：
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作者：
Georgiou, G;Stephens, DL;Earhart, CF
最近已经通过采用融合蛋白方法实现了外源肽和可溶性蛋白的细菌细胞表面暴露。Lpp ′-OmpA（46-159）-Bla融合蛋白先前已经显示在革兰氏阴性细菌大肠杆菌的细胞表面上展示正常周质酶β-内酰胺酶（Bla）。我们研究了三联融合蛋白的OmpA结构域在过客结构域（Bla）的表面展示中的作用，并表征了融合蛋白对外膜的完整性和渗透性的影响，我们发现，除了OmpA（46-159）外，由氨基酸46-66组成的第二个OmpA片段也可以介导Bla在细胞表面的展示。（氨基酸46-84、46-109、46-128、46-141和46-145）将Bla结构域锚定在外膜的周质面上或引起外膜的主要破坏，允许抗体渗透到细胞中，洗涤剂和抗生素的敏感性和周质渗漏试验表明，外膜的渗透性的变化是一个不可避免的后果，显示一个大的周质蛋白的大肠杆菌的表面上。这是关于细胞表面工程可能对细菌外膜的完整性和渗透性的影响的第一个系统性报告。
Bacterial cell-surface exposure of foreign peptides and soluble proteins has been achieved recently by employing a fusion protein methodology, An Lpp'-OmpA(46-159)-Bla fusion protein has been shown previously to display the normally periplasmic enzyme beta-lactamase (Bla) on the cell surface of the Gram-negative bacterium Escherichia coli, Here, we have investigated the role of the OmpA domain of the tripartite fusion protein in the surface display of the passenger domain (Bla) and have characterized the effects of the fusion proteins on the integrity and permeability of the outer membrane, We show that in addition to OmpA(46-159), a second OmpA segment, consisting of amino acids 46-66, can also mediate the display of Bla on the cell surface, Other OmpA domains of various lengths (amino acids 46-84, 46-109, 46-128, 46-141 and 46-145) either anchored the Bla domain on the periplasmic face of the outer membrane or caused a major disruption of the outer membrane, allowing the penetration of antibodies into the cell, Detergent and antibiotic sensitivity and periplasmic leakage assays showed that changes in the permeability of the outer membrane are an unavoidable consequence of displaying a large periplasmic protein on the surface of E.coli. This is the first systematic report on the effects that cell surface engineering may have on the integrity and permeability properties of bacterial outer membranes.