Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, Nanjing, Jiangsu, China.
Mol Biotechnol. 2010 Mar;44(3):198-203. doi: 10.1007/s12033-009-9226-0.
Heparin-binding epidermal growth factor (HB-EGF) can stimulate the division of various cell types and has potential clinical applications that stimulate growth and differentiation. HB-EGF has an EGF-like domain typical of all members of the EGF family. The high expression of active HB-EGF in Escherichia coli has not been successful as the protein contains three intra-molecular disulfide bonds, the same as other members of the EGF super family that are difficult to form correctly in the bacterial intracellular environment. This work fused the non-glycosylated HB-EGF gene with a small ubiquitin-related modifier gene (SUMO) by over-lap PCR. The resulting fusion gene SUMO-HBEGF was highly expressed in BL21(DE3) that the soluble SUMO-HBEGF was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease Ulp1 to obtain the native HB-EGF, which was further purified by Ni-NTA affinity chromatography. MTT assays indicated the purified HB-EGF, as well as SUMO-HBEGF, had mitogenic activity in a dose-dependent manner.
肝素结合表皮生长因子（HB-EGF）可刺激多种细胞类型的分裂，具有刺激生长和分化的潜在临床应用。HB-EGF 具有 EGF 家族所有成员典型的 EGF 样结构域。由于该蛋白含有三个分子内二硫键，与 EGF 超家族的其他成员一样，在细菌细胞内环境中难以正确形成，因此在大肠杆菌中高表达有活性的 HB-EGF 尚未成功。本工作通过重叠 PCR 将非糖基化的 HB-EGF 基因与小泛素相关修饰物基因（SUMO）融合。所得融合基因 SUMO-HBEGF 在 BL21(DE3)中高度表达，可溶性 SUMO-HBEGF 占总细胞蛋白的 30%。融合蛋白通过 Ni-NTA 亲和层析纯化，并用 SUMO 特异性蛋白酶 Ulp1 切割，得到天然 HB-EGF，进一步通过 Ni-NTA 亲和层析纯化。MTT 分析表明，纯化的 HB-EGF 和 SUMO-HBEGF 均具有剂量依赖性的有丝分裂活性。
