RESEARCH LETTER Cloning and characterization ofa haloarchaeal heat shock protein 70 functionally expressed in Escherichia coli Hao Zhang, Lu Lin, Chi Zeng, Ping Shen & Yu-Ping Huang Laboratory of Microbial Genetics, College of Life Sciences, Wuhan University, Wuhan, China Downloaded from https://academic.oup.com/femsle/article/275/1/168/499933 by guest on 01 October 2021 Correspondence: Yu-Ping Huang, Abstract Laboratory of Microbial Genetics, College of Life Sciences, Wuhan University, Wuhan The Hsp70 molecular chaperone machine is constituted by the 70-kDa heat shock 430072, China. Tel.: 186-27-68754533; protein Hsp70 (DnaK), cochaperone protein Hsp40 (DnaJ) and a nucleotide- fax: 186-27-68754833; exchange factor GrpE. Although it is one of the best-characterized molecular e-mail: [email protected] chaperone machines, little is known about it in archaea. A 5.2-kb region contain- ing the hsp70 (dnaK) gene was cloned from Natrinema sp. J7 strain and sequenced. Received 7 May 2007; revised 29 June 2007; It contained the Hsp70 chaperone machine gene locus arranged unidirectionally in accepted 13 July 2007. the order of grpE, hsp70 and hsp40 (dnaJ). The hsp70 gene from Natrinema sp. J7 First published online August 2007. was overexpressed in Escherichia coli BL21 (DE3). The recombinant Hsp70 protein was in a soluble and active form, and its ATPase activity was optimally active in DOI:10.1111/j.1574-6968.2007.00881.x 2.0 M KCl, whereas NaCl had less effect. In vivo, the haloarchaeal hsp70 gene 1 Editor: Marco Moracci allowed an E. coli dnak-null mutant to propagate l phages and grow at 42 C. The results suggested that haloarchaeal Hsp70 should be beneficial for extreme Keywords halophiles survival in low-salt environments. hsp70 ; Hsp70; ATPase activity; haloarchaea. structural homology, Hsp70s are highly specific and the Introduction basis of this functional specificity is not well understood A variety of environmental stresses induce the synthesis of a (Buchberger et al., 1994; James et al., 1997). set of highly conserved proteins called heat shock proteins Hsp70 proteins in bacteria and eukaryotes have been (Hsps). Some Hsps are also molecular chaperones, and play extensively studied (Mayer & Bukau, 2005). However, little important roles in normal cellular physiology and in cell information is known regarding haloarchaeal Hsp70 pro- survival in the face of stress (Ellis & van der Vies, 1991). The teins. An Hsp70 homologue from Halobacterium cutiru- heat shock Hsp70 system is one of the best-characterized brum was identified and purified (Tokunaga et al., 1999). chaperone machineries, which contains Hsp70 (DnaK), its Nevertheless, the functional specificity of haloarchaeal cochaperone protein Hsp40 (DnaJ) and the nucleotide Hsp70 has not been reported. Here, the hsp70 gene was exchange factor GrpE (Bukau & Horwich, 1998). Hsp70 cloned from an extremely halophilic archaeon Natrinema sp. systems are not only universally distributed in bacteria and J7, which harbors a high copy number plasmid pHH205 (Ye eukaryotes but are also found in some archaea, especially et al., 2003) and possesses extracellular proteolytic activity that they occur in all haoarchaea with no exceptions (Shi et al., 2006). Then, the ATPase activities of recombinant (Macario et al., 2004). Hsp70 protein expressed in Escherichia coli and functional Hsp70 proteins are one of the most conserved proteins complementation of an E. coli dnaK mutant were analyzed. known to date, which form the central part of the Hsp70 system. Hsp70s are composed of a highly homologous N-terminal ATPase domain, a substrate-binding domain Materials and methods and a C-terminal variable domain (Mayer & Bukau, 2005), Strains, plasmids and culture conditions the ATPase activities of which are all stimulated by Hsp40, GrpE and substrate binding (Liberek et al., 1991), and the Natrinema sp. J7 strain was grown in a complex medium at molecular chaperone functions of Hsp70 seem to be based 45 1C(Yeet al., 2003). The E. coli MC4100 dnaK-null mutant on its ATPase activity. Despite the high sequence and BM271 (Paek & Walker, 1987) and plasmid pTrc99a (Wang c 2007 Federation of European Microbiological Societies FEMS Microbiol Lett 275 (2007) 168–174 Published by Blackwell Publishing Ltd. All rights reserved A haloarchaeal Hsp70 functionally expressed in E. coli 169 Table 1. The oligodeoxynucleotide PCR primers used in this paper with the DNA probes. The inserted fragments obtained from Primer Nucleotide sequence Reference the selected positive plasmid were sequenced. For the construction of the expression vector, the haloarch- Primer63 50-GGAAGGCAGCGACCCCGAAATC-30 hsp70 Primer964 50-CCTGTGGCATTCTCGTGGACCC-30 primers for aeal hsp70 ORF was amplified by PCR with the primer HKF- Halobacterium NdeI and HKR-HindIII. The PCR fragment was digested with sp. NRC-1 the NdeI and HindIII, and then ligated into NdeI/HindIII- HKF-NdeI 50-GGGCATATGGCGAGCAACAAAATTC-30 hsp70 primers digested pET26b1 to construct plasmid pHK2. HKR-HindIII 50-GGGAAGCTTCTCCTCGTCGTCCTCGTC-30 for Natrinema Another expression plasmid, named pTrcK, was con- sp. J7 structed by the introduction of the NcoI–HindIII full-length KP-RT 50-GTCGGCTCGTTGAT-30 hsp70 specific KPF-Out 50-TACTTCTCCGACCGACAGC-30 primers for ORF of hsp70 into a plasmid pTrc99a previously digested KPR-Out 50-GCGTTTGATCTTCTGGAGGA-30 RLM-RACE PCR with the same enzymes. KPF-Inner 50-CGCATCATCAACGAGCCGA-30 Downloaded from https://academic.oup.com/femsle/article/275/1/168/499933 by guest on 01 October 2021 0 0 KPR-Inner 5 -ATCGTCTTTTCGGGGTTCTG-3 Overexpression, purification and ATPase activity assay of the haloarchaeal Hsp70 The expression of Hsp70 was achieved by induction of the et al., 2004) were kindly gifted by Prof. B. Lipinska˜ (Gdansk˜ cultures with 1 mM isopropyl-b-D-thiogalactopyranoside University, Poland) and Dr M.R. Volkert (University of (IPTG) at A 0.6, followed by an incubation of 12 h at Massachusetts Medicine School), respectively. Plasmid 600 nm 30 1C. Then, cultures were harvested and resuspended in pDNAJ (Yoshimune et al., 2005), an expression plasmid for 20 mM Tris-Cl buffer (pH 7.2). Following cell lysis by E. coli DnaJ (EcoDnaJ), was kindly provided by Dr N. Esaki sonication (3–6 pulses of 1 min, interspersed with cooling (Kyoto University, Japan). on ice), cell debris was pelleted by centrifugation at 12 000 g for 20 min, and clear supernatants were obtained. Molecular genetic methods Purifications of Hsp70 were carried out with the Ni-NTA HisBind affinity chromatography kit according to the The genome DNA and total RNA were isolated and purified method of QIAGEN (2002). The protein concentrations according to protocols described in The Halohandbook: were determined using the Bradford method (Bradford, Protocols for Halobacterial Genetics, version 4.9 (http:// 1976) with bovine serum albumin as a standard, and the www.microbiol.unimelb.edu.au/people/dyallsmith/resources/ homogeneity of the final Hsp70 preparation was confirmed halohandbook/index.html). Molecular cloning techniques by 10% sodium dodecylsulfate-polyacrylamide gel were performed using standard procedures (Sambrook & electrophoresis (SDS-PAGE). Russell, 2001). RNA ligase-mediated rapid amplification of ATPase activity of Hsp70 protein was determined by cDNA ends by PCR (RLM-RACE PCR) was performed using measuring the free phosphate released from the ATP hydro- the 50-full RACE Core Set (Takara Biotech, China) according lysis according to the malachite green method (Lanzetta et al., to the manufacturer’s instructions. The oligodeoxynucleo- 1979). All reactions were performed in buffer M (50 mM Tris- tide primers used in this study are shown in Table 1. Cl, pH 7.6; 10 mM MgCl2;5mMATP),and2MKClwas added when the effects of temperature on the ATPase Gene cloning and plasmids construction activities were studied. The salt dependence of the ATPase activity was measured between 0 and 3 M NaCl or KCl, For Southern blot analysis, the genomic DNA was digested respectively. After a 30-min incubation of Hsp70 with buffer with restriction enzymes and separated by electrophoresis in M, the reaction was stopped by addition of 20% tricarboxylic 1% agarose gel and then transferred to a nylon membrane acid. The standard curve was obtained using phosphate buffer (Hybond-N1, Amersham, Buckinghamshire, UK). The 900- (pH 6.8). The enzymatic activity was calculated as nanomole bp PCR-amplified fragment of hsp70 (positions 63–964) of ATP per milligram of DnaK per minute. from Halobacterium sp. NRC-1 was used as a probe. The digoxigenin-labeled probes were hybridized with the digested Immunological identification of the fragments at 42 1C overnight. The membrane was washed, haloarchaeal recombinant Hsp70 and the hybridized signals were detected with a digoxigenin detection kit (Boehringer Mannheim, Germany) as described In order to exclude the contamination of E. coli endogenous in the supplier’s instructions. The DNA fragments whose chaperones in archaeal Hsp70 preparation, Western blotting sizes were similar to those of the hybridized fragments were was performed by transferring the proteins, after electro- ligated into the plasmid vector pUC18, and the resultant phoresis on 10% acrylamide gels, to a nitrocellulose mem- constructs were introduced into E. coli JM109. The plasmids brane using the Transblot cell from BioRad, and then carrying the hsp70 genes were identified by hybridization incubating the membrane with polyclonal anti-DnaKEc, FEMS Microbiol Lett 275 (2007) 168–174 c 2007 Federation of European Microbiological