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. 2009 Jun 3;49(2):151–160. doi: 10.1007/s12088-009-0029-6

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

Wan-Chi Liang 1, Xuan-Hui Wang 1, Min-Guan Lin 2, Long-Liu Lin 1,✉
PMCID: PMC3450139  PMID: 23100764

Abstract

The heat shock protein 70 (Hsp70/DnaK) gene of Bacillus licheniformis is 1,839 bp in length encoding a polypeptide of 612 amino acid residues. The deduced amino acid sequence of the gene shares high sequence identity with other Hsp70/DnaK proteins. The characteristic domains typical for Hsps/DnaKs are also well conserved in B. licheniformis DnaK (BlDnaK). BlDnaK was overexpressed in Escherichia coli using pQE expression system and the recombinant protein was purified to homogeneity by nickel-chelate chromatography. The optimal temperature for ATPase activity of the purified BlDnaK was 40°C in the presence of 100 mM KCl. The purified BlDnaK had a Vmax of 32.5 nmol Pi/min and a KM of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756-ts mutant to grow at 44°C, suggesting that BlDnaK should be functional for survival of host cells under environmental changes especially higher temperature. We also described the use of circular dichroism to characterize the conformation change induced by ATP binding. Binding of ATP was not accompanied by a net change in secondary structure, but ATP together with Mg2+ and K+ ions had a greater enhancement in the stability of BlDnaK at stress temperatures. Simultaneous addition of DnaJ, GrpE, and NR-peptide (NRLLLTG) synergistically stimulates the ATPase activity of BlDnaK by 11.7-fold.

Keywords: Bacillus licheniformis, DnaK, ATPase activity, Escherichia coli, Circular dichroism

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References

  • 1.Ritossa E. A new puffing pattern induced by heat shock and DNP in Drosophilia. Experientia. 1962;18:571–573. [Google Scholar]
  • 2.Hartl F.U. Molecular chaperones in cellular protein folding. Nature. 1996;381:571–579. doi: 10.1038/381571a0. [DOI] [PubMed] [Google Scholar]
  • 3.Bukau B., Horwich A.L. The Hsp70 and Hsp60 chaperone machines. Cell. 1998;92:351–366. doi: 10.1016/S0092-8674(00)80928-9. [DOI] [PubMed] [Google Scholar]
  • 4.Kregel K.C. Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Physiol. 2002;92:2177–2186. doi: 10.1152/japplphysiol.01267.2001. [DOI] [PubMed] [Google Scholar]
  • 5.Mayer M.P., Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci. 2005;62:670–684. doi: 10.1007/s00018-004-4464-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Liberck K., Marszalek J., Ang D., Georgopoulos S., Zylicz M. Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci USA. 1991;88:2874–2878. doi: 10.1073/pnas.88.7.2874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Cheetham M.E., Caplan A.J. Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell Stress Chaperones. 1998;3:28–36. doi: 10.1379/1466-1268(1998)003<0028:SFAEOD>2.3.CO;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Kelly W.L. Molecular chaperones: how J domains turn on Hsp70s. Curr Biol. 1999;9:R305–R308. doi: 10.1016/S0960-9822(99)80185-7. [DOI] [PubMed] [Google Scholar]
  • 9.Jordan R., McMacken R. Modulation of the ATPase activity of the molecular chaperone DnaK by peptides and the DnaJ and GrpE heat shock proteins. J Biol Chem. 1995;270:4563–4569. doi: 10.1074/jbc.270.9.4563. [DOI] [PubMed] [Google Scholar]
  • 10.Eveleigh D.E. The microbial production of industrial chemicals. Sci Am. 1981;245:155–178. doi: 10.1038/scientificamerican0981-154. [DOI] [Google Scholar]
  • 11.Gupta R., Beg Q.K., Lorenz P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol. 2002;59:13–32. doi: 10.1007/s00253-002-0975-y. [DOI] [PubMed] [Google Scholar]
  • 12.Crabb W.D., Shetty J.K. Commodity scale production of sugars from starches. Curr Opin Microbiol. 1999;2:252–256. doi: 10.1016/S1369-5274(99)80044-7. [DOI] [PubMed] [Google Scholar]
  • 13.Birrer G.A., Cromwick A.M., Gross R.A. γ-Poly(glutamic acid) formation of Bacillus licheniformis 9945a: physiological and biochemical studies. Intl J Biol Macromol. 1994;16:265–275. doi: 10.1016/0141-8130(94)90032-9. [DOI] [PubMed] [Google Scholar]
  • 14.Ming L.J., Epperson J.D. Metal binding and structure-activity relationship of the metalloantibiotic peptide bacitracin. J Inorg Chem. 2002;91:46–58. doi: 10.1016/s0162-0134(02)00464-6. [DOI] [PubMed] [Google Scholar]
  • 15.Murphy T., Roy I., Harrop A., Dixon K., Keshavarz T. Effect of oligosaccharide elicitors on bacitracin A production and evidence of transcriptional level control. J Biotechnol. 2007;131:397–403. doi: 10.1016/j.jbiotec.2007.07.943. [DOI] [PubMed] [Google Scholar]
  • 16.Zuo R. Biofilms: strategies for metal corrosion inhibition employing microorganisms. Appl Microbiol Biotechnol. 2007;76:1245.1253. doi: 10.1007/s00253-007-1130-6. [DOI] [PubMed] [Google Scholar]
  • 17.Schlicker C., Bukau B., Mogk A. Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implication for their applicability in biotechnology. J Biotechnol. 2002;96:13–21. doi: 10.1016/S0168-1656(02)00033-0. [DOI] [PubMed] [Google Scholar]
  • 18.Langer T., Lu C., Echols H., Flanagan J., Hayer M.K., Hartl F.U. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature. 1992;356:683–689. doi: 10.1038/356683a0. [DOI] [PubMed] [Google Scholar]
  • 19.Banecki B., Zylicz M. Real time kinetics of the DnaK/DnaJ/GrpE molecular chaperone machine action. J Biol Chem. 1996;271:6137–6143. doi: 10.1074/jbc.271.11.6137. [DOI] [PubMed] [Google Scholar]
  • 20.Gamber J., Multhaup G., Tomoyasu T., McCarty J.S., Rudiger S., Schonfeld J., Schirra C., Bujard H., Bukau B. A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor σ32. EMBO J. 1996;15:607–617. [PMC free article] [PubMed] [Google Scholar]
  • 21.Gässler C.S., Buchberger A., Laufer T., Mayer M.P., Schroder H., Valencia A., Bukau B. Mutations in the DnaK chaperone afftecting interaction with the DnaJ cochaperone. Proc Natl Acad Sci USA. 1998;95:15229–15234. doi: 10.1073/pnas.95.26.15229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Han W., Christen P. Mechanism of the targeting action of DnaJ in the DnaK molecular chaperone system. J Biol Chem. 2003;278:19038–19043. doi: 10.1074/jbc.M300756200. [DOI] [PubMed] [Google Scholar]
  • 23.Tilly K., Hauser R., Campbell J., Ostheimer G.J. Isolation of dnaJ, dnaK and grpE homologous from Borrella burgdorferi and complementation of Escherichai coli mutants. Mol Microbiol. 1993;7:359.369. doi: 10.1111/j.1365-2958.1993.tb01128.x. [DOI] [PubMed] [Google Scholar]
  • 24.Motohashi K., Yohda M., Endo I., Yoshida M. A novel factor required for the assembly of the DnaK and DnaJ chaperone of Thermus thermophilus. J Biol Chem. 1996;271:17343–17348. doi: 10.1074/jbc.271.29.17343. [DOI] [PubMed] [Google Scholar]
  • 25.Zuber M., Hoover T.A., Dertzbaugh M.T., Court D.L. Analysis of the DnaK molecular chaperone system of Francisella tularensis. Gene. 1995;164:149–152. doi: 10.1016/0378-1119(95)00489-S. [DOI] [PubMed] [Google Scholar]
  • 26.Boshoff A., Hennessy F., Blatch G.L. The in vivo and in vitro characterization of DnaK from Agrobacterium tumefaciens RUOR. Protein Expr Purifi. 2004;38:161–169. doi: 10.1016/j.pep.2004.06.039. [DOI] [PubMed] [Google Scholar]
  • 27.Rupprecht E., Gathmann S., Fuhrmann E., Schneider D. Three different DnaK proteins are functionally expressed in the cyanobacterim Synechocystis sp. PCC 6803. Microbiology. 2007;153:1828.1841. doi: 10.1099/mic.0.2007/005876-0. [DOI] [PubMed] [Google Scholar]
  • 28.Zhang H., Lin L., Zeng C., Shen P., Huang Y.P. Cloning and characterization of a haloarchaeal heat shock protein 70 functionally expressed in Escherichia coli. FEMS Microbiol Lett. 2007;275:168–174. doi: 10.1111/j.1574-6968.2007.00881.x. [DOI] [PubMed] [Google Scholar]
  • 29.Boshoff A., Stephens L.L., Blatch G.L. The Agrobacterium tumefaciens DnaK: ATPase cycle, oligomeric state and chaperone properties. Intl J Biochem Cell Biol. 2008;40:804–812. doi: 10.1016/j.biocel.2007.10.017. [DOI] [PubMed] [Google Scholar]
  • 30.Rey M.W., Ramaiya P., Nelson B.A., Brody-Karpin S.D., Zaretsky E.J., Tang M., Lopez de Leon A., Xiang H., Gusti V., Clausen I.G., Olsen P.B., Rasmussen M.D., Andersen J.T., Joergensen P.L., Laesen T.S., Sorokin A., Bolotin A., Lapidus A., Galleron N., Ehrlich S.D., Berka R.M. Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Genome Biol. 2004;5:R077.1–R077.12. doi: 10.1186/gb-2004-5-10-r77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Doi R.H., Rodriguez R.L., Trait R.C. Recombinant DNA techniques: an introduction. MA, USA: Addison-Wesley; 1983. pp. 162–164. [Google Scholar]
  • 32.Sambrook J., Russell D.W. Molecular cloning: a laboratory manual. 3rd edn. NY, USA: Cold Spring Harbor Laboratory, Cold Spring Harbor; 2001. [Google Scholar]
  • 33.Dagert M., Ehrlich S.D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979;6:23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  • 34.Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  • 35.Bradford M.M. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  • 36.Lanzett P.A., Alvarez L.J., Reinach P.S., Candia O.A. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979;100:95–97. doi: 10.1016/0003-2697(79)90115-5. [DOI] [PubMed] [Google Scholar]
  • 37.Chamberlain L.H., Burgoyne R.D. Ativation of the ATPase activity of heat shock proteins Hsc70 / Hsp70 by cysteine-string protein. Biochem J. 1997;322:853–858. doi: 10.1042/bj3220853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Bohm G., Murh R., Jaenicke R. Quantitative analysis of protein far-UV circular dichroism spectra by neutral networks. Protein Eng. 1992;5:191–195. doi: 10.1093/protein/5.3.191. [DOI] [PubMed] [Google Scholar]
  • 39.Hibion T., Kaku N., Yoshikawa H., Takabe T., Takabe T. Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, and copper binding under various salinity conditions. Plant Mol Biol. 1999;40:409–418. doi: 10.1023/A:1006273124726. [DOI] [PubMed] [Google Scholar]
  • 40.Burkholder W.F., Panagiotidis C.A., Silverstein S.J., Cegielska A., Gottesman M.E., Gaitanaris G.A. Isolation and characterization of an Escherichia coli DnaK mutant with impaired ATPase activity. J Biol Chem. 1994;242:364–377. doi: 10.1006/jmbi.1994.1587. [DOI] [PubMed] [Google Scholar]
  • 41.Kamath-Loeb A.S., Lu C., Suh W., Lonetto M.A., Gross G.A. Analysis of three DnaK mutant proteins suggests that progression through the ATPase cycle requires conformational changes. J Biol Chem. 1995;270:30051–30059. doi: 10.1074/jbc.270.50.30051. [DOI] [PubMed] [Google Scholar]
  • 42.Paek K.H., Walker G.C. Escherichia coli dnaK dull mutants are inviable at high temperatures. J Bacteriol. 1987;169:283–290. doi: 10.1128/jb.169.1.283-290.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Bukau B., Walker G.C. Delta dnak52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal growth temperatures. J Bacteriol. 1989;171:6030–6038. doi: 10.1128/jb.171.11.6030-6038.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Hesterkamp T., Bukau B. Role of the DnaK and HscA homologs of Hsp70 chaperones in protein folding in E.coli. EMBO J. 1998;17:4818–4828. doi: 10.1093/emboj/17.16.4818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Buchberger A., Gassler C.S., Buttner M., McMacken R., Bukau B. Functional defects of the DnaK756 mutant chaperone of Escherichia coli indicate distinct roles for amino- and carboxyl-terminal residues in substrate and co-chaperone interaction and interdomain communication. J Biol Chem. 1999;274:38017–38026. doi: 10.1074/jbc.274.53.38017. [DOI] [PubMed] [Google Scholar]
  • 46.Johnson W.C., Jr. Protein secondary structure and circular dichroism: a practical guide. Proteins. 1990;7:205–214. doi: 10.1002/prot.340070302. [DOI] [PubMed] [Google Scholar]
  • 47.Flynn G.C., Chappell T.G., Rothman J.E. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science. 1989;245:385–390. doi: 10.1126/science.2756425. [DOI] [PubMed] [Google Scholar]
  • 48.Gragerov A., Zeng L., Zhao X., Burkholder W., Gottesman M.E. Specificity of DnaK-peptide binding. J Mol Biol. 1994;235:848–854. doi: 10.1006/jmbi.1994.1043. [DOI] [PubMed] [Google Scholar]
  • 49.Palleros D.R., Reid K.L., Shi L., Welch W.J., Fink A.L. ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis. Nature. 1993;365:664–666. doi: 10.1038/365664a0. [DOI] [PubMed] [Google Scholar]
  • 50.Genevaux P., Georgopoulos C., Kelly W.L. The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions. Mol Microbiol. 2007;66:840–857. doi: 10.1111/j.1365-2958.2007.05961.x. [DOI] [PubMed] [Google Scholar]
  • 51.Palleros D.R., Welch W.J., Fink A.L. Interaction of Hsp70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding. Proc Natl Acad Sci USA. 1991;88:5719–5723. doi: 10.1073/pnas.88.13.5719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Palleros D.R., Reid K.L., McCarty J.S., Walker G.C., Fink A.L. DnaK, Hsp73, and their molten globules: two different ways heat shock proteins respond to heat. J Biol Chem. 1992;276:6098–6104. [PubMed] [Google Scholar]
  • 53.Borges J.C., Ramos C.H.I. Spectroscopic and thermodynamic measurements of nucleotide-induced changes in the human 70-kDa heat shock cognate protein. Arch Biochem Biophys. 2006;452:46–54. doi: 10.1016/j.abb.2006.05.006. [DOI] [PubMed] [Google Scholar]
  • 54.Grimshaw J.P.A., Jelesarov H., Schönfelds H.J., Christen P. Reversible thermal transition in GrpE, the nucleotide exchange factor of the DnaK heat-chock system. J Biol Chem. 2001;276:6098–6104. doi: 10.1074/jbc.M009290200. [DOI] [PubMed] [Google Scholar]
  • 55.Montgomery D.L., Morimoto R.I., Gierasch L.M. Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling. J Mol Biol. 1999;286:915–932. doi: 10.1006/jmbi.1998.2514. [DOI] [PubMed] [Google Scholar]
  • 56.Mayer M.P., Brehmer D., Gässler C.S., Bukau B. Hsp70 chaperone machines. Adv Protein Chem. 2001;50:1–45. doi: 10.1016/S0065-3233(01)59001-4. [DOI] [PubMed] [Google Scholar]
  • 57.Wegele H., Müller L., Bucher J. Hsp70 and Hsp90 — a relay team for protein folding. Rev Physiol Biochem Pharmacol. 2004;151:1–44. doi: 10.1007/s10254-003-0021-1. [DOI] [PubMed] [Google Scholar]
  • 58.Vogel M., Mayer M.P., Bukau B. Allosteric regulation of Hsp7- chaperones involves a conserved interdomain linker. J Biol Chem. 2006;281:38705–38711. doi: 10.1074/jbc.M609020200. [DOI] [PubMed] [Google Scholar]

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