Volume 10, Issue 2 (2019)
JMBS 2019, 10(2): 165-172 |
Back to browse issues page
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Vahdani F, Ghafoori H, Sarikhan S. Sequencing, cloning and expression of DnaK chaperone from Bacillus halodurans Guj1. JMBS 2019; 10 (2) :165-172
URL: http://biot.modares.ac.ir/article-22-15778-en.html
URL: http://biot.modares.ac.ir/article-22-15778-en.html
1- Biology Department, Science Faculty, University of Guilan, Rasht, Iran
2- Biology Department, Science Faculty, University of Guilan, Rasht, Iran, Biology Department, Sciences Faculty, University of Guilan, Namjoo Street, Rasht, Iran. Postal Code: 4193833697 ,h.ghafoori@guilan.ac.ir
3- Molecular Bank, Iranian Biological Resource Center (IBRC), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
2- Biology Department, Science Faculty, University of Guilan, Rasht, Iran, Biology Department, Sciences Faculty, University of Guilan, Namjoo Street, Rasht, Iran. Postal Code: 4193833697 ,
3- Molecular Bank, Iranian Biological Resource Center (IBRC), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
Abstract: (9362 Views)
Hsp70 family members are central components of the cellular network of molecular chaperones and folding catalysts. The gene encoding a protein related to Hsp70 or DnaK in the domain bacteria is called dnaK. DnaK proteins are involved in de novo protein folding, formation, and disassembly of protein complexes and degradation of misfolded proteins. The gene dnaJ which codes for Hsp40 in bacteria, modulate the activities of DnaK by acting as co-chaperone. In the present study, we cloned and expressed DnaK from Bacillus halodurans Guj1 were identified. The dnaK gene of B. halodurans was successfully expressed in E. coli BL21 (DE3) using pET-28a+ expression system. The open reading frame of the cloned gene contained 1839bp and encoded 612 amino acid residues. Calculated molecular weight and pI of the protein were 66.18kDa and 4.55 respectively. The deduced amino acid sequence of B. halodurans Guj1 showed about 60% identity with the E. coli counterpart. The 3D structure of dnaK from B. halodurans was constructed using the crystal structure of human HSP70 chaperone BiP as the template, which showed an identity of 88.8% together. Partially purified recombinant DnaK by heat treatment showed a band at approximately 70kDa on SDS-PAGE. Our findings showed that the recombinant DnaK improved the refolding efficiency of the carbonic anhydrase by 27% after 60min at 54°C. According to the results obtained, DnaK from B. halodurans can potentially be used for improving the functional properties of enzymes and proteins in various applications.
Article Type: Research Paper |
Subject:
Agricultural Biotechnology
Received: 2017/08/12 | Accepted: 2017/11/14 | Published: 2019/06/20
Received: 2017/08/12 | Accepted: 2017/11/14 | Published: 2019/06/20
References
1. Smith HL, Li W, Cheetham ME. Molecular chaperones and neuronal proteostasis. Semin Cell Dev Biol. 2015;40:142-52. [Link] [DOI:10.1016/j.semcdb.2015.03.003]
2. Dobson CM, Šali A, Karplus M. Protein folding: A perspective from theory and experiment. Angew Chem Int Ed Engl. 1998;37(7):868-93.
https://doi.org/10.1002/(SICI)1521-3773(19980420)37:7<868::AID-ANIE868>3.0.CO;2-H [Link] [DOI:10.1002/(SICI)1521-3773(19980420)37:73.0.CO;2-H]
3. Izaddoust Kordmahaleh M, Ghafoori H, Sarikhan S, Heidari B. Identification and sequence analysis of cDNA encoding the 90-kDa heat shock protein (Hsp90) from the Caspian kutum Rutilus frisii kutum. Aquat Physiol Biotechnol. 2017;4(4):1-12. [Persian] [Link]
4. Hartl FU, Bracher A, Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis. Nature. 2011;475(7356):324-32. [Link] [DOI:10.1038/nature10317]
5. Liberek K, Marszalek J, Ang D, Georgopoulos C, Zylicz M. Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci U S A. 1991;88(7):2874-8. [Link] [DOI:10.1073/pnas.88.7.2874]
6. Zhu X, Zhao X, Burkholder WF, Gragerov A, Ogata CM, Gottesman ME, et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science. 1996;272(5268):1606-14. [Link] [DOI:10.1126/science.272.5268.1606]
7. Mayer MP, Bukau B. Hsp70 chaperones: Cellular functions and molecular mechanism. Cell Mol Life Sci. 2005;62(6):670-84. [Link] [DOI:10.1007/s00018-004-4464-6]
8. Rappa F, Farina F, Zummo G, David S, Campanella C, Carini F, et al. HSP-molecular chaperones in cancer biogenesis and tumor therapy: An overview. Anticancer Res. 2012;32(12):5139-50. [Link]
9. Stefani M, Dobson CM. Protein aggregation and aggregate toxicity: New insights into protein folding, misfolding diseases and biological evolution. J Mol Med (Berl). 2003;81(11):678-99. [Link] [DOI:10.1007/s00109-003-0464-5]
10. Yamaguchi H, Miyazaki M. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies. Biomolecules. 2014;4(1):235-51. [Link] [DOI:10.3390/biom4010235]
11. Jia Q, Luo Y. The selective roles of chaperone systems on over-expression of human-like collagen in recombinant Escherichia coli. J Ind Microbiol Biotechnol. 2014;41(11):1667-75. [Link] [DOI:10.1007/s10295-014-1500-x]
12. Nishihara K, Kanemori M, Kitagawa M,Yanagi H, Yura T. Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl Environ Microbiol. 1998;64(5):1694-9. [Link]
13. Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T. Chaperone coexpression plasmids: Differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl Environ Microbiol. 1998;64(5):1694-9. [Link]
14. Ghafoori H, Askari M, Sarikhan S. Molecular cloning, expression and functional characterization of the 40-kDa heat shock protein, DnaJ, from Bacillus halodurans. Process Biochem. 2017;54:33-40. [Link] [DOI:10.1016/j.procbio.2016.12.017]
15. Schlecht R, Erbse AH, Bukau B, Mayer MP. Mechanics of Hsp70 chaperones enables differential interaction with client proteins. Nat Struct Mol Biol. 2011;18(3):345-51. [Link] [DOI:10.1038/nsmb.2006]
16. Bertelsen EB, Chang L, Gestwicki JE, Zuiderweg ER. Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate. Proc Natl Acad Sci U S A. 2009;106(21):8471-6. [Link] [DOI:10.1073/pnas.0903503106]
17. Nicoll WS, Boshoff A, Ludewig MH, Hennessy F, Jung M, Blatch GL. Approaches to the isolation and characterization of molecular chaperones. Protein Expr Purif. 2006;46(1):1-15. [Link] [DOI:10.1016/j.pep.2005.08.005]
18. Yang J, Nune M, Zong Y, Zhou L, Liu Q. Close and allosteric opening of the polypeptide-binding site in a human Hsp70 chaperone BiP. Structure. 2015;23(12):2191-203. [Link] [DOI:10.1016/j.str.2015.10.012]
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |