Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-D-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite, N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other His.

• MeSH
• acetylglukosamin chemie   metabolismus   MeSH
• chromatografie afinitní MeSH
• endotoxiny biosyntéza   izolace a purifikace   MeSH
• Escherichia coli metabolismus   MeSH
• glutaminfruktosa-6-fosfáttransaminasa (izomerizující) biosyntéza   izolace a purifikace   MeSH
• kultivační média chemie   MeSH
• myši MeSH
• proteiny tepelného šoku HSP70 biosyntéza   izolace a purifikace   MeSH
• průmyslová mikrobiologie metody   MeSH
• rekombinantní proteiny biosyntéza   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

The aim of our study was to test the influence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 oC or 42.5 oC) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 oC) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 oC or 42.5 oC) or standard temperature (control 37.5 oC), post-incubated overnight (16-20 h) at 37.5 oC and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 oC did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 oC. Western-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 oC) and embryos (exposed to 41.5 oC). Following the elevation of temperature to 42.5 oC embryo development was dramatically compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis.

• MeSH
• aktiny fyziologie   izolace a purifikace   MeSH
• apoptóza genetika   MeSH
• financování organizované využití   MeSH
• indukovaná hypertermie metody   využití   MeSH
• králíci růst a vývoj   MeSH
• proteiny tepelného šoku HSP70 izolace a purifikace   škodlivé účinky   MeSH
• tvorba embrya pro výzkumné účely metody   MeSH
• western blotting metody   využití   MeSH
• Check Tag
• králíci růst a vývoj   MeSH