Georgiou G, Stephens D L, Stathopoulos C, Poetschke H L, Mendenhall J, Earhart C F
Department of Chemical Engineering, University of Texas, Austin 78712, USA.
Protein Eng. 1996 Feb;9(2):239-47. doi: 10.1093/protein/9.2.239.
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.
最近，通过采用融合蛋白方法，已实现了外源肽和可溶性蛋白在细菌细胞表面的展示。先前已证明，一种Lpp'-OmpA(46 - 159)-Bla融合蛋白可在革兰氏阴性细菌大肠杆菌的细胞表面展示通常位于周质的酶β-内酰胺酶（Bla）。在此，我们研究了三方融合蛋白的OmpA结构域在乘客结构域（Bla）表面展示中的作用，并表征了融合蛋白对细菌外膜完整性和通透性的影响。我们发现，除了OmpA(46 - 159)之外，由氨基酸46 - 66组成的第二个OmpA片段也能介导Bla在细胞表面的展示。不同长度的其他OmpA结构域（氨基酸46 - 84、46 - 109、46 - 128、46 - 141和46 - 145）要么将Bla结构域锚定在外膜的周质面上，要么导致外膜的严重破坏，使抗体能够进入细胞。去污剂和抗生素敏感性以及周质渗漏试验表明，外膜通透性的变化是在大肠杆菌表面展示大型周质蛋白不可避免的结果。这是关于细胞表面工程可能对细菌外膜完整性和通透性特性产生影响的第一份系统性报告。
