PTD-hEGF融合蛋白在大肠杆菌中的表达及活性分析

PTD-hEGF Fusion Protein—Gene Expression and Function Analysis

摘要:

为获得hEGF在大肠杆菌中的高效表达，构建了pPTD-hEGF原核表达载体。将其转化大肠杆菌BL21（DE3），经0.3mmol/L IPTG诱导，PTD-hEGF融合蛋白进行了有效表达，表达产物主要形成了包涵体。经Ni²⁺-NTA亲和层析纯化，融合蛋白的纯度达99.5%。MALDI-TOF-MS分析证明表达的融合蛋白氨基酸序列完全正确。PTD-hEGF融合蛋白能明显促进HEK-293细胞的分裂和生长。

关键词:PTD-hEGF融合蛋白，原核表达，HEK-293细胞

Abstract:

To produce membrane-permeable human epidermal growth factor (hEGF)，a pPTD-hEGF prokaryocyte expression vector was constructed and transformed into E. coli BL 21 (DE3). The PTD-hEGF fusion protein was induced by 0.3mmol/L of IPTG expressed in the form of inclusion body with an yield of 40% of the total protein in the cells，and then purified by Ni²⁺-NTA affinity chromatography. The SDS-PAGE analysis showed a single fusion protein band with a molecular weight of 16kD. The amino acid sequence was checked by MALDI-TOF-MS analysis. The genetic engineering PTD-hEGF fusion protein obviously promoted the proliferation and growth of the HEK-293 cells in vitro.

Key words:PTD-hEGF fusion protein，prokaryocyte expression，HEK-293 cell