Janpan Piyapon, Schmelzer Bernhard, Klamrak Anuwatchakij, Tastub Patthana, Upathanpreecha Tewa, Rahman Shaikh Shahinur, Nabnueangsap Jaran, Saengkun Yutthakan, Rungsa Prapenpuksiri, Mattanovich Diethard, Daduang Sakda
Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40000, Thailand.
J Fungi (Basel). 2024 Dec 11;10(12):854. doi: 10.3390/jof10120854.
Hyaluronidases have been a subject of great interest in medical and cosmeceutical applications. Previously, our group demonstrated that the venom glands of contain hyaluronidase enzymes (VesT2s), and heterologous expression of the corresponding gene () in systems results in inclusion bodies, necessitating functional folding using urea. Here, we report the successful heterologous expression of VesT2a in the expression system, with gene construction achieved using Golden. After confirming gene integration in the yeast genome, methanol-induced cultures yielded an exceptional amount of VesT2a, approximately two-fold higher than that obtained with the constitutive expression vector (P). Upon culturing in a bioreactor, yeast cells harboring pAOX1-αMF- produced secreted proteins with a total yield of 96.45 mg/L. The secreted VesT2a has a molecular weight of 59.35 kDa, significantly higher than the expected molecular weight (~40.05 kDa), presumably due to endogenous glycosylation by the yeast cells. It exhibits optimal activity at 37 °C and pH 3, showing a specific activity of 4238.37 U/mg, and remains active across a broad range of pH and temperature. Notably, it exhibits higher hyaluronidase activity than the crude venom and -expressed protein, likely due to improved folding via endogenous post-translational modifications, such as disulfide bonds and N-glycosylation; this underscores the potential of heterologous systems for producing venomous hyaluronidases from other species. In silico docking-based analyses further support its catalytic activity and provide insights into seeking natural inhibitors from phenolic-rich plant extracts to alleviate symptoms in patients suffering from insect bites and stings.
透明质酸酶一直是医学和药妆应用领域备受关注的对象。此前，我们团队证明，[具体物种]的毒腺含有透明质酸酶（VesT2s），在[表达系统名称]系统中对相应基因（[基因名称]）进行异源表达会产生包涵体，需要使用尿素进行功能折叠。在此，我们报告了VesT2a在[表达系统名称]表达系统中的成功异源表达，基因构建使用了Golden技术。在确认基因整合到酵母基因组后，甲醇诱导培养产生了大量的VesT2a，比组成型表达载体（P）获得的产量高出约两倍。在生物反应器中培养时，携带pAOX1-αMF-[相关基因]的酵母细胞分泌的蛋白质总产量为96.45 mg/L。分泌的VesT2a分子量为59.35 kDa，显著高于预期分子量（约40.05 kDa），这可能是由于酵母细胞的内源性糖基化作用。它在37°C和pH 3时表现出最佳活性，比活性为4238.37 U/mg，并且在广泛的pH和温度范围内都保持活性。值得注意的是，它表现出比粗毒液和[表达系统名称]表达的蛋白质更高的透明质酸酶活性，这可能是由于通过内源性翻译后修饰（如二硫键和N-糖基化）改善了折叠；这凸显了异源系统生产其他物种有毒透明质酸酶的潜力。基于计算机对接的分析进一步支持了其催化活性，并为从富含酚类的植物提取物中寻找天然抑制剂以减轻昆虫叮咬和蜇伤患者的症状提供了见解。
