葡萄糖激酶的异源表达及葡萄糖激酶激动剂筛选体系的建立

Heterologous expression of glucokinase and establishment of a glucokinase agonist screening system

目的葡萄糖激酶(GK)是治疗Ⅱ型糖尿病的重要靶标。旨在通过原核表达获得人源GK,并建立葡萄糖激酶激动剂(GKAs)分子筛选体系,用于筛选靶向GK的抗Ⅱ型糖尿病先导化合物。方法采用基因重组的方法构建GK原核重组表达载体GK-pET28a,将其转化入感受态细胞BL21(DE3)中,采用IPTG诱导重组蛋白的表达,且对诱导条件进行优化。使用Ni-NTA对融合蛋白进行分离纯化。通过Western-blot鉴定蛋白且通过双酶偶联法对蛋白酶进行活力测试。建立葡萄糖激酶激动剂分子筛选体系,筛选活性化合物,并通过表面等离子共振技术(SPR)和计算机分子模拟技术探究活性化合物与葡萄糖激酶的结合情况。结果通过基因重组将葡萄糖激酶与pET28a原核表达载体构建成融合质粒,并且确定最佳诱导表达条件为:0.5 mmol·L^−1,37℃,4~5 h。最佳纯化条件为:含10 mmol·L^−1咪唑缓冲液洗杂蛋白,含100 mmol·L^−1咪唑缓冲液洗脱目的蛋白。通过Western-Blot鉴定表达的蛋白为目的蛋白,且蛋白酶具有较高的活力。成功建立GKAs分子筛选体系。利用SPR技术确定活性化合物与GK有较强的结合,并采用分子对接对其结合位点进行了预测。

Objective Glucokinase(GK)is an important target for the treatment of type 2 diabetes.The aim is to obtain human-derived GK through prokaryotic expression,and establish the glucokinase agonist(GKAs)molecular screening system for anti-type 2 diabetes lead compounds targeting the GK.Methods GK prokaryotic recombinant expression vector GK-pET28a was constructed by genetic recombination,and transformed into competent cell BL21(DE3).The expression of recombinant protein was induced by IPTG,and the induction conditions were optimized.Next,Ni-NTA was used to isolate and purify the fusion protein.Proteins were identified by Western-blot and protease activity tests were performed by a dual enzyme coupling method.Finally,a glucokinase agonist molecular screening system was established for active compounds,and the binding of active compounds to glucokinase was investigated by surface plasmon resonance(SPR)and computer molecular simu-lation techniques.Results GK and pET28a prokaryotic expression vectors were constructed into fusion plasmids by genetic recombination,and determined the best inducible expression conditions:0.5 mmol·L^−1,37℃,4~5 h.The optimal purification conditions were:washing protein with 10 mmol·L^−1 imidazole buffer,and eluting the target protein with 100 mmol·L^−1 imidazole buffer.The expressed protein was identified as the target protein by Western-blot,and the protease had higher activity.A molecular screening system for glucokinase agonists was established successfully.Finally,the strong binding of the active compound to GK was determined using SPR technology,and the binding site was predicted using molecular docking.