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, H6NtHSP70-2 (hexahistidine-tagged Nicotiana tabacum heat shock protein 70-2), maintained the stability of E. coli proteins up to 90°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°C) for recombinant H6NtHSP70-2, E. coli cells overexpressing H6NtHSP70-2 survived about seven times longer than those lacking H6NtHSP70-2. After 2 h at 50°C, only the E. coli overexpressing H6NtHSP70-2 survived under such conditions. Our NtHSP70-2 bioassays, as well as in vitro studies, strongly suggest that HSP70 confers thermo-tolerance to E. coli.