Department of Biological Science and Technology, Tokushima University Graduate School, Minamijosanjima-cho, Tokushima, 770-8506, Japan.
Department of Biological Science and Technology, Tokushima University Graduate School, Minamijosanjima-cho, Tokushima, 770-8506, Japan.
Biochem Biophys Res Commun. 2021 Apr 16;549:105-112. doi: 10.1016/j.bbrc.2021.02.096. Epub 2021 Mar 2.
Escherichia coli is one of the most popularly used hosts to produce recombinant proteins. Most recombinant proteins are produced in the cytoplasm and periplasm, requiring multiple steps to extract and purify recombinant proteins. The Serratia marcescens Lip system (LipB-LipC-LipD) is a type 1 secretion system that selectively secretes LipA from the intracellular to extracellular space in a single step. This study aimed to establish a secretory production system for nanobodies, camelid-derived small molecule antibody fragments, using the S. marcescens Lip system. Surprisingly, E. coli harboring only LipC, a membrane fusion protein of the Lip system, could secrete an anti-green fluorescent protein (GFP)-Nb, a nanobody against GFP, without the addition of a long amino acid sequence. The LipC-based secretion system recognized the Val-Thr-Val sequence at the C-terminus of the nanobody. Finally, Strep-tagged anti-GFP-Nb was purified from culture supernatants of E. coli harboring LipC by Strep-affinity chromatography at a final yield of >5 mg per liter of culture supernatant. These results potently supported that the S. marcescens LipC-based secretion system has the potential to establish an efficient secretory production system for nanobodies.
大肠杆菌是最常用的宿主之一，用于生产重组蛋白。大多数重组蛋白在细胞质和周质中产生，需要经过多个步骤才能提取和纯化重组蛋白。粘质沙雷氏菌 Lip 系统（LipB-LipC-LipD）是一种 1 型分泌系统，可将 LipA 从细胞内一次性选择性分泌到细胞外空间。本研究旨在利用粘质沙雷氏菌 Lip 系统建立纳米抗体（骆驼科来源的小分子抗体片段）的分泌生产系统。令人惊讶的是，仅含有 LipC（Lip 系统的膜融合蛋白）的大肠杆菌可以分泌抗绿色荧光蛋白（GFP）的纳米抗体（针对 GFP 的纳米抗体），而无需添加长的氨基酸序列。LipC 基分泌系统识别纳米抗体 C 末端的 Val-Thr-Val 序列。最后，通过 Strep 亲和层析从含有 LipC 的大肠杆菌的培养上清液中纯化 Strep 标记的抗 GFP-Nb，最终产量超过每升培养上清液 5 毫克。这些结果有力地支持了粘质沙雷氏菌 LipC 基分泌系统有潜力建立高效的纳米抗体分泌生产系统。
