Luo Xue-Gang, Ma De-Yun, Wang Yue, Li Wen, Wang Chong-Xi, He Ying-Ying, Gu Xiang-Chao, Li Xiu-Mei, Zhou Hao, Zhang Tong-Cun
a Key Lab of Industrial Fermentation Microbiology (Tianjin University of Science and Technology) , Ministry of Education , Tianjin , P.R. China.
b Tianjin Key Lab of Industrial Microbiology , College of Biotechnology, Tianjin University of Science and Technology , Tianjin , P.R. China.
Biosci Biotechnol Biochem. 2016;80(3):584-90. doi: 10.1080/09168451.2015.1091714. Epub 2015 Oct 7.
Administration of macromolecule compositions in medicine and cosmetics always exhibited low bioavailability due to the limitation of transmembrane transport. Here, human epidermal growth factor (hEGF) was fused with glutathione S-transferase (GST) and Pep-1, the first commercial cell-penetrating peptide, in Escherichia coli. The fusion protein was firstly purified with the affinity chromatography, and then the GST tag was released by TEV protease. Final purification was achieved by the ion exchange chromatography. The biological activities and the transmembrane ability of the obtained products were determined using scratch wound-healing assay, MTT analysis, and immunofluorescence assay. The results showed that both rhEGF and Pep-1-fused hEGF were soluble expressed in E. coli. The fusion of Pep-1 could markedly increase the transmembrane ability of EGF, whereas it did not interfere with the growth-stimulating and migration-promoting functions of hEGF on fibroblasts. This research provided a novel strategy for the transmembrane transport of protein-derived cosmetics or drugs.
由于跨膜运输的限制，在医学和化妆品中施用大分子组合物时，其生物利用度一直较低。在此，人表皮生长因子（hEGF）与谷胱甘肽S-转移酶（GST）以及首个商业化的细胞穿透肽Pep-1在大肠杆菌中进行融合。首先通过亲和色谱法纯化融合蛋白，然后用TEV蛋白酶释放GST标签。最终通过离子交换色谱法实现纯化。使用划痕伤口愈合试验、MTT分析和免疫荧光试验测定所得产物的生物活性和跨膜能力。结果表明，rhEGF和Pep-1融合的hEGF均在大肠杆菌中可溶性表达。Pep-1的融合可显著提高EGF的跨膜能力，而不干扰hEGF对成纤维细胞的生长刺激和迁移促进功能。本研究为蛋白质类化妆品或药物的跨膜运输提供了一种新策略。
