GSK, via Fiorentina 1, 53100, Siena, Italy.
Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.
Microb Cell Fact. 2021 Feb 2;20(1):33. doi: 10.1186/s12934-021-01528-z.
The display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins.
In this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp'OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp'OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37 and 25 °C as demonstrated by FACS analysis, confocal and transmission electron microscopy.
To our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp'OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.
在细胞表面展示重组蛋白在疫苗开发、肽文库筛选、全细胞生物催化剂以及用于诊断、工业或环境目的的生物传感器开发等方面具有广泛的应用。在过去的几十年中，已经开发了多种表面展示系统，用于在大肠杆菌表面展示重组蛋白，例如自转运蛋白和外膜蛋白。
在这项研究中，我们评估了三种在大肠杆菌表面展示一组异源和同源成熟脂蛋白的方法：来自脑膜炎奈瑟菌的四种和来自宿主菌株的四种，已知这些蛋白位于外膜的内叶。构建了携带编码八种成熟脂蛋白序列的构建体，每个融合到三个不同系统的输送部分：来自肠致病性大肠杆菌的弥漫性粘附-I （DAI-I）相关的自转运蛋白粘附素、Lpp'OmpA 嵌合体和来自丁香假单胞菌的冰核蛋白（INP）的截断形式 InaK-NC（N 端域与 C 端域融合）。与 INP 构建体观察到的情况相反，当与 AIDA-I 或 Lpp'OmpA 融合时，大多数成熟脂蛋白在 37 和 25°C 下都能在细菌表面显示出来，这通过 FACS 分析、共聚焦和透射电子显微镜证明。
据我们所知，这是第一项使用多种载体蛋白比较表面展示系统的研究。我们表明，实验条件，包括载体蛋白的选择和生长温度，在成熟脂蛋白转移到细菌表面方面起着重要作用。尽管使用 InaK-NC 锚定基序进行了所有优化步骤，但在本研究中使用的载体蛋白的表面暴露并未实现。对于我们的实验条件，Lpp'OmpA 嵌合体已被证明是同源载体蛋白的有效表面展示系统，尽管观察到细胞裂解和表型异质性。最后，AIDA-I 被发现是大肠杆菌宿主菌株中成熟脂蛋白（特别是异源蛋白）的最佳表面展示系统，没有生长抑制，只有有限的表型异质性。
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