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首页> 外文期刊>Applied Microbiology >Identification of Candidate Carrier Proteins for Surface Display on Lactococcus lactis by Theoretical and Experimental Analyses of the Surface Proteome
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Identification of Candidate Carrier Proteins for Surface Display on Lactococcus lactis by Theoretical and Experimental Analyses of the Surface Proteome

机译：通过表面蛋白质组学的理论和实验分析鉴定在乳酸乳球菌表面展示的候选载体蛋白

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Lactococcus lactis is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of L. lactis MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of L. lactis NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in L. lactis NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.
机译：乳酸乳球菌是一种经证实可用于人类口腔应用的安全性的乳酸菌。为此目的，重组蛋白的表面展示是重要的，并且正在寻找用于其的新方法。细菌表面蛋白质组的分析对于识别用于表面展示的新候选载体蛋白至关重要。我们通过使用Augur和LocateP软件对乳酸乳球菌MG1363的表面相关蛋白做出了两种不同的预测，分别产生了666和648个蛋白。通过使用蛋白质组学方法鉴定了乳酸乳球菌NZ9000（MG1363的衍生物）的表面蛋白。从完整细菌上切下表面蛋白，并通过质谱鉴定所得的肽。后一种方法产生了80种蛋白质，其中任何一种软件均未预测其中的34种。在80种蛋白质中，选择了7种进行进一步研究。将它们与C-末端六组氨酸标签按框克隆，并使用乳链菌肽控制的表达在乳酸乳球菌NZ9000中过表达。检测到带有六组氨酸标签的正确分子量的蛋白质。通过流式细胞仪确认其表面定位。碱性膜蛋白A（BmpA）暴露在最高水平。为了测试BmpA作为候选载体蛋白，在基因构建体中将六组氨酸标签替换为葡萄球菌蛋白A的B结构域。 B结构域以BmpA为载体显示在表面上。证明了共价BmpA结合的优势。因此，在乳球菌表面展示中显示出BmpA是载体蛋白的合适候选者。

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