title: ATP-independent thermoprotective activity of Nicotiana tabacum heat shock protein 70 in Escherichia coli.
abstract: To study the functioning of HSP70 in Escherichia coli, we selected NtHSP70-2 (AY372070) from among three genomic clones isolated in Nicotiana tabacum. Recombinant NtHSP70-2, containing a hexahistidine tag at the amino-terminus, was constructed, expressed in E. coli, and purified by Ni(2+) affinity chromatography and Q Sepharose Fast Flow anion exchange chromatography. The expressed fusion protein, H(6)NtHSP70-2 (hexahistidine-tagged Nicotiana tabacum heat shock protein 70-2), maintained the stability of E. coli proteins up to 90 degrees C. Measuring the light scattering of luciferase (luc) revealed that NtHSP70-2 prevents the aggregation of luc without ATP during high-temperature stress. In a functional bioassay (1 h at 50 degrees C) for recombinant H(6)NtHSP70-2, E. coli cells overexpressing H(6)NtHSP70-2 survived about seven times longer than those lacking H(6)NtHSP70-2. After 2 h at 50 degrees C, only the E. coli overexpressing H(6)NtHSP70-2 survived under such conditions. Our NtHSP70-2 bioassays, as well as in vitro studies, strongly suggest that HSP70 confers thermo-tolerance to E. coli.
authors.affiliation: Department of Bio-Food Materials, College of Medical Life Science, Silla University, Busan 617-736, Korea. choeun@silla.ac.kr
authors.forename: Eun Kyung
authors.initials: EK
authors.lastname: Cho
authors.forename: Song-Ja
authors.initials: SJ
authors.lastname: Bae
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology*; D002470:Cell Survival; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008156:Luciferases / Q000032:analysis; D018832:Molecular Chaperones / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D014026:Nicotiana / Q000235:genetics*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D008156:Luciferases