Department of Life Sciences, National Chung Hsing University, No. 145, Xing-Da Road, South District, Taichung City, 40227, Taiwan, ROC.
Section of Neurology, Department of Internal Medicine, Tungs' Taichung Metro-Harbor Hospital, No. 699, Section 8, Taiwan Boulevard, Wuqi District, Taichung City, 43503, Taiwan, ROC.
Appl Microbiol Biotechnol. 2024 Oct 28;108(1):496. doi: 10.1007/s00253-024-13336-7.
Melittin is a bioactive peptide and the predominant component in bee venom (BV), studied for its many medical properties, such as antibacterial, anti-inflammatory, anti-arthritis, nerve damage reduction, and muscle cell regeneration. Melittin is primarily obtained through natural extraction and chemical synthesis; however, both methods have limitations and cannot be used for mass production. This study established a heterologous melittin expression system in the probiotic yeast Kluyveromyces marxianus. This yeast was selected for its advantages in stress tolerance and high secreted protein yields, simplifying purification. A > 95% high-purity melittin (MET) and its precursor promelittin (ProMET) were successfully produced and purified at 1.68 μg/mL and 3.33 μg/mL concentrations and verified through HPLC and mass spectrum. The functional test of the NSC-34 cell regeneration revealed that MET achieved the best activity compared to ProMET and the natural-extracted BV groups. Growth-related gene expressions were evaluated, including microtubule-associated protein 2 (MAP2), microtubule-associated protein Tau (MAPT), growth-associated protein 43 (GAP-43), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and acetylcholine esterase (AChE). The results indicated that treating MET increased MAP2, GAP-43, and VAChT expressions, in which cholinergic signaling is related to neurological functions. A heterologously expressed melittin in a probiotic yeast and its potential for promoting NSC-34 regeneration described here facilitate commercial and therapeutic use. KEY POINTS: • MET and its precursor ProMET were successfully hetero-expressed in K. marxianus • > 95% high-purity MET and ProMET were purified at 1.68 μg/mL and 3.33 μg/mL • MET has no cytotoxicity toward NSC-34 and significantly promotes NSC-34 growth.
蜂毒素是一种生物活性肽，也是蜂毒液 (BV) 的主要成分，因其许多医学特性而受到研究，如抗菌、抗炎、抗关节炎、减少神经损伤和肌肉细胞再生。蜂毒素主要通过天然提取和化学合成获得；然而，这两种方法都有其局限性，不能用于大规模生产。本研究在益生菌酵母克鲁维酵母中建立了一种异源蜂毒素表达系统。选择这种酵母是因为它具有应激耐受和高分泌蛋白产量的优势，简化了纯化过程。以 >95%的高纯度蜂毒素 (MET) 和其前体预蜂毒素 (ProMET) 分别在 1.68 μg/mL 和 3.33 μg/mL 浓度下成功生产和纯化，并通过 HPLC 和质谱验证。对 NSC-34 细胞再生的功能测试表明，与 ProMET 和天然提取的 BV 组相比，MET 表现出最佳活性。评估了与生长相关的基因表达，包括微管相关蛋白 2 (MAP2)、微管相关蛋白 Tau (MAPT)、生长相关蛋白 43 (GAP-43)、胆碱乙酰转移酶 (ChAT)、囊泡乙酰胆碱转运体 (VAChT) 和乙酰胆碱酯酶 (AChE)。结果表明，用 MET 处理可增加 MAP2、GAP-43 和 VAChT 的表达，其中胆碱能信号与神经功能有关。本文描述的在益生菌酵母中异源表达蜂毒素及其促进 NSC-34 再生的潜力，有利于商业和治疗用途。关键点：• MET 和其前体 ProMET 成功在 K. marxianus 中异源表达• 以 >95%的纯度在 1.68 μg/mL 和 3.33 μg/mL 下纯化 MET 和 ProMET• MET 对 NSC-34 无细胞毒性，并显著促进 NSC-34 生长。
