重组蜘蛛丝蛋白MiSp NT结构域在不同pH环境下的Trp荧光光谱及圆二色谱分析

为明确蛛丝蛋白NT结构域的生物学功能，首次研究了MiSp NT结构域在不同pH条件下的二聚化动力学及二级结构特性. 基于蛛丝蛋白MiSp全长编码基因，克隆并分别在BL21(DE3)和Rosetta-gami 2(DE3)中重组表达MiSp NT结构域: BL21(DE3)表达水平较高，约35 mg/L LB培养基，表达产物需经短暂超声和2 mol/L尿素促溶；Rosetta-gami 2(DE3)表达产物可溶性较好，但表达水平仅为BL21 (DE3) 一半左右. 融合蛋白经凝血酶介导的2次Ni-NTA纯化，纯度达到95%以上. Trp 荧光光谱分析表明,MiSpNT在pH为7.0左右时开始二聚化，pH5.7时二聚体为主要构象，pH_T约为6.6. MiSp NT在pH 7.5，6.5和5.5条件下的CD图谱相似，均主要为α-helix，表明NT二聚化与二级结构水平的变化没有直接联系. 本研究为进一步探索蛛丝蛋白NT结构域的结构和功能以及成丝机理提供线索.

Trp Fluorescence and CD Spectra of Recombinant MiSp NT Protein of Spider Silk under Different pH Conditions

To find out the biological functions of spidroin NT domains, dimerization dynamics and characterization of secondary structures under different pH based on MiSp NT domain were analyzed. According to the full-length MiSp gene sequence already obtained, the NT domain gene fragment was cloned and expressed via DNA recombinant technique at the first time in BL21(DE3) and Rosetta-gami 2(DE3), respectively. The expression level of Bl21(DE3) could go up to 35 mg/L LB medium which is about two fold higher than that of Rosetta-gami 2(DE3). However, recombinant proteins from Rosetta-gami 2(DE3) were soluble while the proteins from BL21(DE3) were mainly in the pellet fragment which could have a good solubility after treated with 2 mol/L urea and short time sonication. The MiSp NT proteins were purified twice by Ni-NTA mediated by thrombin cleavage which gave a purity of >95%. MiSpNT proteins can be dimeric with pH below 7.0 and will mainly adopt dimer conformation when the pH is below 5.7 indicated by Trp fluorescence, and the transition pH is about 6.6. Under pH 7.5, 6.5 and 5.5,CD spectra showed the predominant secondary structure of MiSp NT was α-helix, and under different pH values,no dramatic changes happened which suggested no relationship between NT dimerization and obvious secondary structure changes. Our study is helpful to determine the NT domain structure and functions, and also spidroin-silk formation mechanism.