State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
Appl Environ Microbiol. 2010 Apr;76(8):2410-8. doi: 10.1128/AEM.01752-09. Epub 2010 Feb 19.
The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage phiPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli. After being mixed with various cells in vitro, GFP was successfully displayed on the surfaces of Lactococcus lactis, Lactobacillus casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, beta-galactosidase (beta-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and beta-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB.
来自乳酸乳球菌温和噬菌体 phiPYB5 的溶菌酶 Lyb5 对革兰氏阳性菌和革兰氏阴性菌表现出广泛的溶菌谱。序列分析表明，溶菌酶 Lyb5（Ly5C）的 C 末端包含三个推定的溶菌酶基序（LysM）重复区域，这意味着 Ly5C 参与了细菌细胞壁的结合。为了研究 Ly5C 用于表面展示的潜力，将绿色荧光蛋白（GFP）融合到 Ly5C 的 N 或 C 末端，并在大肠杆菌中表达所得融合蛋白。在体外与各种细胞混合后，GFP 成功地显示在乳球菌、干酪乳杆菌、短乳杆菌、植物乳杆菌、发酵乳杆菌、德氏乳杆菌、乳双歧杆菌和嗜热链球菌细胞的表面上。与未经预处理的细胞相比，经化学预处理的乳球菌和干酪乳杆菌细胞的荧光强度增加表明肽聚糖是 Ly5C 的结合配体。此外，优化了 GFP-Ly5C 的结合能力的 pH 值和氯化钠浓度，并在最佳条件下观察到细胞的高强度荧光。所有结果表明，Ly5C 是构建乳酸细菌（LAB）表面展示系统的新型锚定蛋白。为了证明 Ly5C 介导的表面展示系统的适用性，用 GFP 替换来自芽孢杆菌属 K1 菌株的β-半乳糖苷酶（β-Gal），通过 Ly5C 功能性地展示在 LAB 细胞的表面上。GFP 和β-Gal 的表面展示成功为噬菌体溶菌酶细胞壁锚定蛋白在 LAB 中的表面展示开辟了可行性。
