细胞穿膜肽与人表皮生长因子在大肠杆菌中的重组融合表达

Recombinant Fusion Expression of Cell-penetrating Peptide and Human Epidermal Growth Factor in Escherichia coli

摘要:

该研究构建了细胞穿膜肽Pep-1和人表皮生长因子hEGF融合基因（epEGF）的重组表达质粒PGEX-4T-1-epEGF，并成功转化大肠杆菌BL21（DE3）得到重组大肠杆菌株BL21（DE3）/PGEX-4T1-epEGF。通过IPTG诱导发酵，制备重组融合人表皮生长因子蛋白，并初步尝试建立纯化研究，有效实现了人表皮生长因子（hEGF）在大肠杆菌中的可溶性表达。该研究通过发酵条件优化，发现发酵过程中温度、诱导剂浓度、诱导时间、培养基都是影响蛋白表达量的关键因素；温度在20 ℃、IPTG浓度0.4 mmol/L、诱导时间8 h、培养基为TB液体培养基，目的蛋白占总蛋白的量达到21.58%，蛋白浓度为0.13 mg/mL。菌体破碎液上清经过GST亲和层析融合蛋白纯度达到66.82%，阴离子交换层析后融合蛋白纯度达到92.57%。该研究成功在大肠杆菌中表达出可溶形式的hEGF，且纯化工艺较简单，为hEGF的进一步开发利用提供了参考。

Abstract:

In this study, the recombinant expression plasmid PGEX-4T-1-epEGF containing the fusion genes of cell-penetrating peptide Pep-1 and human epidermal growth factor hEGF (epEGF) was constructed and successfully transformed into E. coli BL21 (DE3) to obtain a recombinant E coli BL21 (DE3)/ PGEX-4T1-epEGF. The recombinant fusion human epidermal growth factor was prepared through IPTG induced fermentation, the preliminary purification was established, and the soluble expression of human epidermal growth factor (hEGF) in E. coli was effectively achieved. Through optimization of fermentation conditions, it was found that temperature, inducer concentration, induction time and culture medium were the key factors affecting protein expression; Under the conditions of 20 ℃, IPTG concentration of 0.4 mmol/L, induction time of 8 h, and use of TB liquid medium, the target protein accounted for 21.58% of the total protein and the protein concentration was 0.13 mg/mL. The purities of the fusion protein after GST affinity chromatography and anion exchange chromatography were 66.82% and 92.57%, respectively. In this study, the soluble form of hEGF was successfully expressed in E. coli, and the purification process was relatively simple, which provided a reference for the further exploitation and utilization of hEGF.