Molecular Medicine Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
Department of Biotechnology, School of Advanced Medical Science and Technologies, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Curr Pharm Biotechnol. 2021;22(9):1192-1205. doi: 10.2174/1389201021666201012162904.
The selection of a suitable signal peptide that can direct recombinant proteins from the cytoplasm to the extracellular space is an important criterion affecting the production of recombinant proteins in Escherichia coli, a widely used host. Nanobodies are currently attracting the attention of scientists as antibody alternatives due to their specific properties and feasibility of production in E. coli.
CD44 nanobodies constitute a potent therapeutic agent that can block CD44/HA interaction in cancer and inflammatory diseases. This molecule may also function as a drug against cancer cells and has been produced previously in E. coli without a signal peptide sequence. The goal of this project was to find a suitable signal peptide to direct CD44 nanobody extracellular secretion in E. coli that will potentially lead to optimization of experimental methods and facilitate downstream steps such as purification.
We analyzed 40 E. coli derived signal peptides retrieved from the Signal Peptide database and selected the best candidate signal peptides according to relevant criteria including signal peptide probability, stability, and physicochemical features, which were evaluated using signalP software version 4.1 and the ProtParam tool, respectively.
In this in silico study, suitable candidate signal peptide(s) for CD44 nanobody secretory expression were identified. CSGA, TRBC, YTFQ, NIKA, and DGAL were selected as appropriate signal peptides with acceptable D-scores, and appropriate physicochemical and structural properties. Following further analysis, TRBC was selected as the best signal peptide to direct CD44 nanobody expression to the extracellular space of E. coli.
The selected signal peptide, TRBC is the most suitable to promote high-level secretory production of CD44 nanobodies in E. coli and potentially will be useful for scaling up CD44 nanobody production in experimental research as well as in other CD44 nanobody applications. However, experimental work is needed to confirm the data.
选择合适的信号肽，使其能够将重组蛋白从细胞质导向细胞外空间，是影响大肠杆菌等广泛应用宿主中重组蛋白生产的重要标准。由于具有独特的特性和在大肠杆菌中生产的可行性，纳米抗体目前正作为抗体替代品吸引着科学家的关注。
CD44 纳米抗体是一种有效的治疗剂，可阻断癌症和炎症性疾病中的 CD44/HA 相互作用。该分子也可能作为抗癌药物发挥作用，之前曾在大肠杆菌中无信号肽序列的情况下生产过。本项目的目的是找到一种合适的信号肽，以指导 CD44 纳米抗体在大肠杆菌中的细胞外分泌，这可能会优化实验方法，并为下游步骤（如纯化）提供便利。
我们分析了从信号肽数据库中检索到的 40 个大肠杆菌衍生的信号肽，根据相关标准（包括信号肽概率、稳定性和理化特性）选择了最佳候选信号肽，分别使用 SignalP 软件版本 4.1 和 ProtParam 工具进行评估。
在这项计算机研究中，确定了适合 CD44 纳米抗体分泌表达的合适候选信号肽。CSGA、TRBC、YTFQ、NIKA 和 DGAL 被选为具有可接受的 D 分数、合适的理化和结构特性的合适信号肽。进一步分析后，TRBC 被选为指导 CD44 纳米抗体在大肠杆菌细胞外空间表达的最佳信号肽。
所选信号肽 TRBC 最适合促进 CD44 纳米抗体在大肠杆菌中的高水平分泌生产，并且可能对实验研究中 CD44 纳米抗体生产的放大以及其他 CD44 纳米抗体应用有用。但是，需要进行实验工作来验证数据。
