重组角质细胞生长因子(KGF)的原核表达及优化

Expression and Optimization of Recombinant Keratinocyte Growth Factor(KGF) in E.coli

[目的]在原核表达系统中进行重组人角质细胞生长因子(KGF)的表达及优化。[方法]选取缺失N端23个氨基酸残基的KGF片段作为目的基因,按照大肠杆菌的密码子偏好性,将其进行密码子优化。使用BL21(DE3)菌株表达KGF(ΔN23)、SUMO融合的SUMOKGF(ΔN23)和Fh8-SUMO融合的Fh8-SUMO-KGF(ΔN23),并测试不同表达温度(37、23℃)对KGF(ΔN23)表达结果的影响。[结果]在BL21(DE3)菌株中,37℃表达时KGF(ΔN23),SUMO-KGF(ΔN23)和Fh8-SUMO-KGF(ΔN23)的表达量都较高,其中可溶性蛋白表达量KGF(ΔN23)<SUMO-KGF(ΔN23)<Fh8-SUMO-KGF(ΔN23)。降低诱导温度为23℃后,3种KGF的表达量都显著降低,但是可溶性蛋白的占比显著提高,均接近100%。[结论]降低诱导温度能够促进KGF的可溶性表达,但是总蛋白的表达水平显著下降;与可溶性标签蛋白融合表达能够提高KGF在37℃表达时可溶性蛋白占比,其中融合Fh8-SUMO标签的KGF表达量和可溶性蛋白占比较高。

[Objective]To express and optimize recombinant human KGF in prokaryotic expression system.[Method]KGF fragment with N-terminal 23 amino acid residues deleted(KGFΔN23)was selected as the target gene,and its codon usage was optimized according to the codon preference of E.coli.We used BL21(DE3)strain to express KGF(N23),SUMO-fused SUMO-KGF(N23)and Fh8-SUMO-fused Fh8-SUMO-KGF(N23),and the effects of different expression temperatures(37,23℃)were tested.[Result]The expressions of KGF(N23),SUMO-KGF(N23)and Fh8-SUMO-KGF(N23)were high in BL21(DE3)strains at 37℃,and the proportion of soluble KGF protein was KGF(ΔN23)<SUMO-KGF(ΔN23)<Fh8-SUMO-KGF(ΔN23).When at 23℃,the expressions of the three KGFs decreased significantly,but the proportion of soluble proteins increased significantly,all approaching 100%.[Conclusion]Decreasing the induction temperature promotes the soluble expression of KGF,but the total expression level decreased significantly.Fusion expression with soluble tag protein increases the proportion of soluble protein when expressed at 37℃,the total expression and soluble expression level of Fh8-SUMO fused KGF were high.