双叉犀金龟表皮蛋白TdCPR12611与TdCPR7854的表达纯化及特性分析

【目的】探析双叉犀金龟Trypoxylus dichotomus表皮蛋白的序列特征及生化性质。【方法】利用RT-PCR克隆双叉犀金龟表皮蛋白基因，利用生物信息学方法分析表皮蛋白的结构特征及系统发育；采用大肠杆菌Escherichia coli表达系统对双叉犀金龟表皮蛋白进行重组表达，并通过金属离子鳌合层析的方法对重组蛋白进行纯化；采用体外结合实验检测双叉犀金龟表皮蛋白与α-几丁质(α-chitin)、β-几丁质(β-chitin)、壳聚糖(chitosan)和胶体几丁质(colloidal chitin)的结合能力；观察确定蛋白液液相分离(liquid-liquid phase separation, LLPS)性质。【结果】克隆获得双叉犀金龟表皮蛋白基因TdCPR12611(GenBank登录号： MT813021)和TdCPR7854(GenBank登录号： MT813022)。系统进化分析结果表明，TdCPR12611与食粪金龟Onthophagus taurus OtCP-1的亲缘关系最近；TdCPR7854与食粪金龟的OtCP-acp20-1, OtCP-acp20-2和OtCP-acp20-3的亲缘关系最为接近，它们均属于CPR_RR-2家族。原核表达和纯化获得重组表皮蛋白TdCPR12611和TdCPR7854。两重组表皮蛋白与4种类型的几丁质具有不同结合能力，其中有41.4%的TdCPR12611与壳聚糖结合，而有62.3%的TdCPR7854与β-几丁质结合。TdCPR12611具有内部较为无序的结构，并能够在室温条件下自发团聚形成液液相分离现象，而TdCPR7854不能。【结论】本研究测定分析了双叉犀金龟CPR_RR-2家族表皮蛋白TdCPR12611与TdCPR7854的序列特征和与几丁质的结合特性。研究结果有利于加深人们对于昆虫表皮装配机制的了解，为蛋白仿生材料开发提供思路。

Expression,purification and characterization of the cuticular proteins TdCPR12611 andTdCPR7854 fromTrypoxylus dichotomus(Coleoptera: Scarabaeidae)

【Aim】 To explore the sequence characteristics and biochemical properties ofcuticular proteins from Trypoxylus dichotomus. 【Methods】 RT-PCR was used to clonecuticular protein genes of T. dichotomus, and the structural features and phylogeny ofcuticular proteins were analyzed by bioinformatics methods. Recombinant cuticular proteinsof T. dichotomus were expressed in Escherichia coli expression system, and purified bymetal-chelating affinity chromatography. In vitro binding experiments were performed todetect the binding ability of cuticular proteins of T. dichotomus with chitins including α-chitin, β-chitin, chitosan and colloidal chitin. Liquid-liquid phase separation (LLPS)was observed and determined. 【Results】 Two cuticular protein genes TdCPR12611 (GenBankaccession no.: MT813021) and TdCPR7854 (GenBank accession no.: MT813022) of T. dichotomuswere cloned and obtained. Phylogenetic analysis results showed that TdCPR12611 is closelyrelated to OtCP-1 from Onthophagus taurus, while TdCPR7854 is closely related toOtCP-acp20-1, OtCP-acp20-2, and OtCP-acp20-3 from O. taurus, all of which belong tothe CPR_RR-2 family. Recombinant TdCPR12611 and TdCPR7854 proteins were expressed byprokaryotic expression and purified. The two recombinant proteins had different bindingabilities with four types of chitins, among which 41.4% of TdCPR12611 could bind withchitosan, while 62.3% of TdCPR7854 could bind with β-chitin. TdCPR12611 was predicted tohave a relatively disordered structure and could spontaneously coacervate at roomtemperature to form liquid-liquid phase separation, while TdCPR7854 could not.【Conclusion】 In this study we assayed and analyzed the sequence characteristics andchitin binding properties of two RR-2 cuticular proteins, TdCPR7854 and TdCPR12611 of T.dichotomus. The results not only deepen our understanding of insect cuticle assemblymechanism, but also provide ideas for the development of protein biomimetic materials.