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...

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.