title: The development of modified human Hsp70 (HSPA1A) and its production in the milk of transgenic mice.
abstract: 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.
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.|Institute of Experimental Cardiology, Cardiology Research Center, Moscow, 125552, Russia.
authors: Yaroslav G
authors: YG
authors: Gurskiy
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
authors: David G
authors: DG
authors: Garbuz
authors: Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.
authors: Nataliya V
authors: NV
authors: Soshnikova
authors: Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.
authors: Aleksey N
authors: AN
authors: Krasnov
authors: Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.
authors: Alexei
authors: A
authors: Deikin
authors: Institute of Cytology, Russian Academy of Sciences, 194064, St. Petersburg, Russia.
authors: Vladimir F
authors: VF
authors: Lazarev
authors: Institute of Cytology, Russian Academy of Sciences, 194064, St. Petersburg, Russia.
authors: Dmitry
authors: D
authors: Sverchinskyi
authors: Institute of Cytology, Russian Academy of Sciences, 194064, St. Petersburg, Russia.
authors: Boris A
authors: BA
authors: Margulis
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
authors: Olga G
authors: OG
authors: Zatsepina
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
authors: Vadim L
authors: VL
authors: Karpov
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
authors: Svetlana N
authors: SN
authors: Belzhelarskaya
authors: Institute of Experimental Cardiology, Cardiology Research Center, Moscow, 125552, Russia.
authors: Evgenia
authors: E
authors: Feoktistova
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.|Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.
authors: Sofia G
authors: SG
authors: Georgieva
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia. misha672011@yahoo.com.|Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow, Russia. misha672011@yahoo.com.
authors: Michael B
authors: MB
authors: Evgen'ev
mesh_terms: D000818:Animals; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000633:toxicity; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008822:Mice, Transgenic; D008892:Milk / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D058849:Protein Refolding; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D018840:HSP70 Heat-Shock Proteins; C490786:HSPA1A protein, human; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins
keywords: Escherichia coli; Glycosylation; Heat shock protein 70; Site-directed mutagenesis; Transgenic mice