Analysis of variants biological function of recombinant human SCF 1-165 and SCF1-141 produced by the yeast expression system

• Main Authors: Ya-Li Tsai, 蔡雅吏
• Other Authors: Yui-Ping Weng
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/36529373714462730078

碩士 === 中華醫事科技大學 === 生物醫學研究所 === 99 === Abstract Human stem cell factor (hSCF), as a glycoprotein, can be widely used in basic and clinical research, biotechnology and the development of industrial processes. It has been shown that SCF initiated diverse cellular responses including hematopoiesis, organgenesis, melanin pigmentation, and development of proliferation, differentiation, migration and survival of cells. To further explore the relationship between structure and function of SCF, we produced rhSCF1-165 (wild type) or rhSCF1-141 (truncated) by yeast expression system and compared their differences between their biological activity and thermal stability. Recombinant hSCF1-141, which is truncated form of rhSCF1-165 in its C-terminus (STLSPEKDSRVSVTKKPFMLPPVA), is the receptor binding domain of rhSCF1-165. Both of them are soluble-type stem cell factor (sSCF). In this study, we showed that overexpression of rhSCF1-165 or rhSCF1-141 can be successfully performed by yeast expression systems in a large amount. In the purification processes of rhSCF, we use hydrophobic interaction chromatography and anion exchange resin chromatography. The biological function study of rhSCF1-165 or rhSCF1-141 was measured by using Alamar blue in TF-1 cell survival assay. We found that it has a dose-dependent proliferation manner at the different concentrations of rhSCF1-141 or rhSCF1-165. At the concentration of 100 ng/ml and different temperatures (25, 50, 70, 90, 110 ℃) under the conditions of the heat 10 minutes, we can observed that the biological activity of rhSCF1-165 are better than rhSCF1-141. Moreover, we found that the biological activity of rhSCF1-165 is higher than rhSCF1-141 when they were heated at 90 ℃ for 0, 2, 10 or 30 minutes at the concentration of 100 ng/ml under the different heating time conditions for 0, 2, 10, 30, 60, or 150 minutes. If the determination of rhSCF1-141 or rhSCF1-165 both at different temperatures (25, 50, 70, 90 ℃) and different concentrations (500, 100, 10 ng/ml) after heating 10 minutes, it showed that both has decline in their biological activity but still be higher than control group in high concentration (500, 100 ng/ml) and under different temperature condition(25, 50, 70, 90 ℃).