PelA-Expansin融合蛋白原核重组表达研究

Prokaryotic expression of PelA-Expansin fusion protein

扩张蛋白(Expansin)具有削弱细胞壁多糖之间氢键,增强多糖降解酶降解多糖的效率。在食品领域、生物能源领域和生物制药领域,具有广泛的应用价值。自然状态下,Expansin蛋白在植物、真菌、细菌等生物中的表达量较少,目前,枯草芽孢杆菌Expansin重组表达研究受到广泛关注。以来源于构巢曲霉的果胶酸裂解酶(PelA)为融合标签,构建高效表达融合蛋白PelA-Expansin的工程菌newexpansin/pelA-pET-28a(+)/BL21(DE3),在OD_(600)为0.4、0.01 mmol/L IPTG、15℃和150 r/min下,表达24 h,融合蛋白产量约为3.0 mg/mL。建立融合蛋白一次性His-tag亲和纯化工艺,工艺中上样、清洗和洗脱缓冲液中咪唑浓度分别为30、60和500 mmol/L,目标蛋白纯度约为95%,得率约为75%;融合蛋白协同纤维素酶、木聚糖酶和果胶酸水解酶酶降解多糖的效率分别为300%、150%和125%;为扩张蛋白Expansin在食品、能源、医药及农业等领域的应用奠定了基础。

Expansin can weaken the hydrogen bond between cell wall polysaccharides and enhance the efficiency of polysaccharide degradation by polysaccharide degrading enzyme.It has a wide range of application value in the field of food,bioenergy and biopharma-ceutical.In the natural state,the expression of expansin protein in plants,fungi,bacteria and other organisms is less.At present,the research on the recombinant expression of expansin in Bacillus subtilis has been widely concerned.In this project,the expression project strain(newexpansin/pelA-pET-28a(+)/BL21(DE3))was constructed with the pectate lyase A(pelA gene from Aspergillus nidulans)as the fusion tag.At OD_(600)=0.4,0.01 mmol/L IPTG,15℃and 150 r/min for 24 h,the fusion protein production was about 3.0 mg/mL.A one-step his-tag affinity purification process was established for the fusion protein and the concentration of imidaz-ole in the sample loading,cleaning and elution buffer were 30,60 and 500 mmol/L,the purity of the target protein was about 95%,and the yield was about 75%.The degradation efficiencies of the fusion protein with cellulase,pectinase and xylanase were 300%,150%and 125%,respectively.This project laid the foundation for the application of expansin in the fields of food,energy,medicine and agriculture.