The development of modified human Hsp70 (HSPA1A) and its production in the milk of transgenic mice

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• 作者：Yaroslav G. Gurskiy ; David G. Garbuz ; Nataliya V. Soshnikova…
• 关键词：Heat shock protein 70 ; Transgenic mice ; Glycosylation ; Site ; directed mutagenesis ; Escherichia coli
• 刊名：Cell Stress and Chaperones
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：21
• 期：6
• 页码：1055-1064
• 全文大小：1,632 KB
• 刊物主题：Biomedicine general; Cell Biology; Biochemistry, general; Immunology; Cancer Research; Neurosciences;
• 出版者：Springer Netherlands
• ISSN：1466-1268
• 卷排序：21

文摘

The production of major human heat shock protein Hsp70 (HSPA1A) in a eukaryotic expression system is needed for testing and possible medical applications. In this study, transgenic mice were produced containing wild-type human Hsp70 allele in the vector providing expression in the milk. The results indicated that human Hsp70 was readily expressed in the transgenic animals but did not apparently preserve its intact structure and, hence, it was not possible to purify the protein using conventional isolation techniques. It was suggested that the protein underwent glycosylation in the process of expression, and this quite common modification for proteins expressed in the milk complicated its isolation. To check this possibility, we mutated all presumptive sites of glycosylation and tested the properties of the resulting modified Hsp70 expressed in E. coli. The investigation demonstrated that the modified protein exhibited all beneficial properties of the wild-type Hsp70 and was even superior to the latter for a few parameters. Based on these results, a transgenic mouse strain was obtained which expressed the modified Hsp70 in milk and which was easy to isolate using ATP columns. Therefore, the developed construct can be explored in various bioreactors for reliable manufacture of high quality, uniform, and reproducible human Hsp70 for possible medical applications including neurodegenerative diseases and cancer.