蜂王浆主蛋白1低聚体的分离纯化及其圆二色谱分析

Purification and Circular Dichroism Analysis of MRJP1 Oligomer from Royal Jelly

蜂王浆主蛋白1(major royal jelly protein 1,MRJP1)是蜂王浆主蛋白家族中最重要的成员之一,MRJP1存在单体和低聚体两种形式。为了研究MRJP1低聚体的二级结构信息,首先利用ToyoScreen GigaCap Q-650M离子交换柱从新鲜蜂王浆中纯化出MRJP1低聚体,经分析超速离心和高效液相色谱-串联质谱技术鉴定其为MRJP1低聚体后,再通过圆二色谱技术检测MRJP1低聚体在不同浓度Ca2+条件下(10、50、200 mmol/L)二级结构的变化情况,并测定其变性温度。结果表明:该分离方法可以一步纯化出MRJP1低聚体,MRJP1低聚体的二级结构中β-折叠占比最高(55%左右),其次是无规卷曲(20%左右)和α-螺旋(15%左右),β-转角(10%左右)含量最低;MRJP1低聚体的变性温度为55℃。实验提出了MRJP1低聚体新的纯化方法及其二级结构的基本信息。

MRJP1 is one of the most important members in the Major Royal Jelly Proteins(MRJPs) family with monomeric and oligomeric forms in royal jelly. In order to investigate the secondary structure of RJP1 oligomer, MRJP1 oligomer was extracted from fresh royal jelly and purified by ToyoScreen GigaCap Q-650 M anion exchange column, then identified by sedimentation equilibrium and liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS). Afterwards, we detected MRJP1 oligomer by synchrotron radiation circular dichroism(SRCD) to clarify the correlation between its secondary structure and different concentration of Ca2+(10, 50 and 200 mmol/L). In addition, its Tm value was also determined. The results showed that the proportion of β-strand(about 55%) was the highest, followed in decreasing order by random coil(20%) and α-helix(15%) and β-turns(10%). Ca2+ had little effect on the secondary structure of MRJP1 oligomer. The Tm value of MRJP1 oligomer was 55 ℃. To conclude, a new method for the purification of MRJP1 oligomer and information on its secondary structure have been presented in this study.