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.

Interplay between recombinant Hsp70 and proteasomes: proteasome activity modulation and ubiquitin-independent cleavage of Hsp70

“…Hsp70 128 has mutations in five putative N-glycosylation sites. However, the protein shows intact chaperone activities and as a wild-type Hsp70 effectively protects cells from ROS (Gurskiy et al 2016). We demonstrated that although much slower than the activated, nonactivated 20S proteasome degraded Hsp70 (Fig.…”

“…In our research, we used wild-type human recombinant Hsp70 (HSPA1A) and modified human Hsp70 with mutated five putative N-glycosylation sites (BHsp70 128^) described in (Gurskiy et al 2016). Sequences encoding both proteins (modified and wild type) were cloned into pET-14b plasmid t o p r o v i d e t h e N -t e r m i n a l p o l y h i s t i d i n e t a g (MGSSHHHHHHSSGLVPRGSH).…”

“…1a). Purity check of Hsp70 128 is described in (Gurskiy et al 2016). Additionally, protein purity was tested by Western blot (data not shown).…”

“…The specific activity of both proteins was measured in vitro. The ability of proteins to reduce endotoxin-induced ROS production was estimated; substrate-binding test and protein substrate refolding test were performed as described in (Gurskiy et al 2016). According to the experiments, both proteins were effective in all the tests described above (data not shown).…”

The Role of p53 Protein in the Realization of the Exogenous Heat Shock Protein 70 Anti-Apoptotic Effect during Axotomy

“…The recombinant human Hsp70 included five substitutions in potential glycosylation sites (N35D, S153A, S362A, T419A, and T489A), which were generated experimentally to reduce protein aggregation and to purify the protein from the milk of transgenic animals [ 19 ]. In previous studies [ 19 ], we have shown that this protein retains all of the biochemical properties (ATP-ase and chaperone activity) inherent to the wild-type protein, but it exhibits a significant reduction of aggregation, which facilitates its purification and increases the immunoregulatory activity. The coding sequence of Hsp70 was cloned into a pET-14b-derived plasmid, in order to add an N-terminal polyhistidine tag (MGSSHHHHHHSSGLVPRGSH).…”