Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
School of Minerals Processing and Bioengineering, Central South University, Changsha, Hunan, 410083, PR China.
Microb Cell Fact. 2024 Apr 12;23(1):108. doi: 10.1186/s12934-024-02386-1.
Microbial cell surface display technology allows immobilizing proteins on the cell surface by fusing them to anchoring motifs, thereby endowing the cells with diverse functionalities. However, the assessment of successful protein display and the quantification of displayed proteins remain challenging. The green fluorescent protein (GFP) can be split into two non-fluorescent fragments, while they spontaneously assemble and emit fluorescence when brought together through complementation. Based on split-GFP assembly, we aim to: (1) confirm the success display of passenger proteins, (2) quantify the number of passenger proteins displayed on individual cells.
In this study, we propose two innovative methods based on split-green fluorescent protein (split-GFP), named GFP1-10/GFP11 and GFP1-9/GFP10-11 assembly, for the purpose of confirming successful display and quantifying the number of proteins displayed on individual cells. We evaluated the display efficiency of SUMO and ubiquitin using different anchor proteins to demonstrate the feasibility of the two split-GFP assembly systems. To measure the display efficiency of functional proteins, laccase expression was measured using the split-GFP assembly system by co-displaying GFP11 or GFP10-11 tags, respectively.
Our study provides two split-GFP based methods that enable qualitative and quantitative analyses of individual cell display efficiency with a simple workflow, thus facilitating further comprehensive investigations into microbial cell surface display technology. Both split-GFP assembly systems offer a one-step procedure with minimal cost, simplifying the fluorescence analysis of surface-displaying cells.
微生物细胞表面展示技术通过将蛋白质融合到锚定基序上，将其固定在细胞表面，从而赋予细胞多种功能。然而，成功展示蛋白质的评估和展示蛋白质的定量仍然具有挑战性。绿色荧光蛋白（GFP）可以分裂成两个非荧光片段，而当它们通过互补自发组装在一起时会发出荧光。基于 GFP 的分裂，我们旨在：（1）确认乘客蛋白的成功展示，（2）量化单个细胞上展示的乘客蛋白数量。
在这项研究中，我们提出了两种基于分裂绿色荧光蛋白（split-GFP）的创新方法，分别命名为 GFP1-10/GFP11 和 GFP1-9/GFP10-11 组装，用于确认成功展示和量化单个细胞上展示的蛋白质数量。我们使用不同的锚定蛋白评估了 SUMO 和泛素的展示效率，以证明两种 GFP 分裂组装系统的可行性。为了测量功能性蛋白质的展示效率，通过共展示 GFP11 或 GFP10-11 标签，使用 GFP 分裂组装系统测量了漆酶的表达。
我们的研究提供了两种基于 GFP 的方法，可通过简单的工作流程对单个细胞的展示效率进行定性和定量分析，从而促进对微生物细胞表面展示技术的进一步全面研究。两种 GFP 分裂组装系统都提供了一步程序，成本最低，简化了表面展示细胞的荧光分析。
