Department of Microbiology, University College Cork, Cork, Ireland.
Microb Cell Fact. 2008 Jul 29;7:24. doi: 10.1186/1475-2859-7-24.
Listeria monocytogenes is a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactis NZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter.
The constructed vector (pNZPnisA:CYTO-LLO) was expressed in L. lactis NZ9000 and was best induced at mid-log phase with 0.2% v/v nisin for 4 h statically at 30 degrees C. Purification of the His-tagged LLO was accomplished by Ni-NTA affinity chromatography and functionality was confirmed through haemolytic assays. Total LLO yield (measured as total protein content) was 4.43-5.9 mg per litre culture and the haemolytic activity was still detectable after 8 months of storage at 4 degrees C.
The LLO production method described in this work provides an approach to efficient LLO production in the Gram-positive Lactococcus bacterium to yield a significant source of the protein for research and diagnostic applications. Expression of LLO in L. lactis has a number of benefits over E. coli which may facilitate both in vivo and in vitro applications of this system.
李斯特菌是一种特征明确的食源性病原体，可感染孕妇和免疫功能低下者。溶血素 O（LLO）是该病原体的主要毒力因子，常用于检测李斯特菌的诊断标志物。此外，LLO 是感染期间驱动 T 细胞介导免疫的有效抗原。在本工作中，乳球菌 NZ9000 被用作表达宿主，通过 NICE（Nisin 控制表达）系统在诱导条件下大量生产 LLO。我们构建了一个经过修饰的 pNZ8048 载体，在强诱导型 PnisA 启动子下游编码了一个六组氨酸标记的 LLO。
构建的载体（pNZPnisA:CYTO-LLO）在乳球菌 NZ9000 中表达，在对数中期用 0.2%v/v 乳链菌肽静态诱导 4 小时，30°C 下诱导效果最佳。通过 Ni-NTA 亲和层析纯化 His 标记的 LLO，并通过溶血实验证实其功能。每升培养物的总 LLO 产量（以总蛋白含量计）为 4.43-5.9mg，4°C 下储存 8 个月后仍可检测到溶血活性。
本工作中描述的 LLO 生产方法提供了一种在革兰氏阳性乳球菌中高效生产 LLO 的方法，为研究和诊断应用提供了大量的蛋白质来源。LLO 在乳球菌中的表达优于大肠杆菌，这可能有利于该系统的体内和体外应用。
