Eglin C与2709碱性蛋白酶相互作用分析及亲和纯化方法

Eglin C是来自水蛭中的一种小型热稳定蛋白质，属于马铃薯胰凝乳蛋白酶抑制剂家族，可以抑制弹性蛋白酶、枯草杆菌蛋白酶、组织蛋白酶、α-lytic蛋白酶以及胰凝乳蛋白酶等。然而，利用eglin C纯化蛋白酶，尚未见研究报道。本文将化学合成的编码 eglin C及其突变体的基因序列，克隆到原核表达载体pQE30，在大肠杆菌BL21获得His6-Tag-eglin C及其突变体的重组蛋白质。SDS-PAGE显示，eglin C蛋白的分子量大约8 kD。His6-Tag-eglin C对胰凝乳蛋白酶、地衣芽孢杆菌2709碱性蛋白酶、枯草芽孢杆菌PB92碱性蛋白酶、枯草杆菌中性蛋白酶的半抑制剂浓度（IC50）分别为0.20±0.15、0.24±0.19、3.33±0.47和52.46±0.38 μmol/L。利用分子对接、基因突变以及荧光光谱等，分析eglin C及其突变体与2709蛋白酶的相互作用。结果显示，2709碱性蛋白酶对eglin C荧光淬灭属于静态淬灭，解离常数为2.60×10-7 mol/L，eglin C中的Asn50 残基对eglin C和2709碱性蛋白酶的结合发挥重要作用。利用eglin C与蛋白酶的特异结合的特性，构建亲和纯化载体，用于纯化来源于地衣芽孢杆菌的2709碱性蛋白酶，相比常规的蛋白酶纯化，显著简化了操作步骤。

Analysis of the Interaction Between Eglin C and 2709 Alkaline Protease and Establishment of an Affinity Purification Method

Eglin C is a small heat-stabilized protein from leeches. It belongs to the potato chymotrypsin inhibitor family, and can inhibit elastase, subtilisin, cathepsin, α-lytic protease and chymotrypsin etc. However, purification of protease by eglin C has not been studied. In this paper, the cDNA sequences encoding eglin C and its mutants were chemically synthesized and cloned into the prokaryotic expression vector pQE30. His6-Tag-eglin C and its mutant recombinant protein were obtained from E. coli BL21 cells. The molecular weight of eglin C is about 8 kD in SDS-PAGE. The IC50 values for His6-Tag-eglin C inhibitory activities for chymotrypsin, Bacillus licheniformis 2709 alkaline protease, Bacillus subtilis PB92 alkaline protease, and Bacillus subtilis neutral protease are: 0.20±0.15, 0.24±0.19, 3.33±0.47, 52.46±0.38 μmol/L, respectively. The interaction of eglin C and its mutants with 2709 protease was evaluated using molecular docking, gene mutation and fluorescence spectroscopy analysis techniques. The results revealed that the quenching of eglin C fluorescence by 2709 alkaline protease was static quenching, with a dissociation constant of 2.60×10^{- 7} mol/L.The Asn50 residue in eglin C plays an important role in the interaction between eglin C and 2709 alkaline protease. In addition, Ni ²⁺-Sepharose 4B-His6-Tag-eglin C are used to purify 2709 alkaline protease from Bacillus licheniformis with the simplest operation compared with conventional protease purification.