Huang Zhifeng, Ni Chunyan, Chu Yanhui, Wang Shanshan, Yang Shulin, Wu Xiaoping, Wang Xiaojie, Li Xiaokun, Feng Wenke, Lin Shaoqiang
Key Laboratory of Biotechnology and Pharmaceutical Engineering of Zhejiang Province, Wenzhou Medical College, Wenzhou 325035, China.
J Biotechnol. 2009 Jul 15;142(3-4):242-9. doi: 10.1016/j.jbiotec.2009.05.004. Epub 2009 May 27.
Recombinant human keratinocyte growth factor 2 (rhKGF-2) is a member of fibroblast growth factor protein family currently being investigated for its promising significant effects in treating epithelial damage. Molecular modification with polyethylene glycol (PEGylation) is an effective approach to improve protein biostability and decrease protein immunogenic activity. In this study, we modified rhKGF-2 through PEGylation at N-terminal residue using 20 kDa PEG-phenyl-isothiocyanate (PIT-PEG20K). PEGylated rhKGF-2 is then purified to near homogeneity by Sephadex G-25 gel filtration followed by a Heparin Sepharose TM CL-6B affinity chromatography. This PEGylated rhKGF-2 retained about 60% of mitogenic activity compared to the non-modified rhKGF-2. Its relative thermal stability at normal physiological temperature and structural stability were significantly enhanced. Moreover, the immunogenicity of PEGylated rhKGF-2 in mice is significant decreased compared to non-modified rhKGF-2. These results suggest that PEGylation of rhKGF-2 could be a more effective approach to the pharmacological and therapeutic application of rhKGF-2.
重组人角质形成细胞生长因子2（rhKGF-2）是成纤维细胞生长因子蛋白家族的一员，目前正在研究其在治疗上皮损伤方面的显著效果。用聚乙二醇进行分子修饰（聚乙二醇化）是提高蛋白质生物稳定性和降低蛋白质免疫活性的有效方法。在本研究中，我们使用20 kDa聚乙二醇-苯基异硫氰酸酯（PIT-PEG20K）对rhKGF-2的N端残基进行聚乙二醇化修饰。然后通过Sephadex G-25凝胶过滤，再经肝素琼脂糖凝胶CL-6B亲和层析，将聚乙二醇化的rhKGF-2纯化至接近均一。与未修饰的rhKGF-2相比，这种聚乙二醇化的rhKGF-2保留了约60%的促有丝分裂活性。其在正常生理温度下的相对热稳定性和结构稳定性显著增强。此外，与未修饰的rhKGF-2相比，聚乙二醇化的rhKGF-2在小鼠体内的免疫原性显著降低。这些结果表明，rhKGF-2的聚乙二醇化可能是rhKGF-2药理和治疗应用的更有效方法。
