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

Liang, Wan-Chi; Wang, Xuan-Hui; Lin, Min-Guan; Lin, Long-Liu

doi:10.1007/s12088-009-0029-6

Cited by 6 publications

(8 citation statements)

References 53 publications

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“…CD spectrum analysis of M. haemofelis rDnaK revealed two minima (at 208 and 222 nm), suggesting that it consisted mostly of ␣-helices (8), which is in good agreement with known DnaK structures, e.g., of E. coli (Protein Database identiy [PDB ID]: 2KH0) and G. kaustophilus (PDB ID: 2V7Y). The structure profile of M. haemofelis rDnaK was insensitive to a change in the presence of nucleotide, as has been shown before for Bacillus licheniformis DnaK (13), and also in the presence of K ϩ and Mg 2ϩ ions. The thermal denaturation of M. haemofelis rDnaK was characterized by two temperature transitions.…”

Section: Discussionsupporting

confidence: 76%

Identification, Characterization, and Application of a Recombinant Antigen for the Serological Investigation of Feline Hemotropic Mycoplasma Infections

Wolf-Jäckel

Jäckel

Museux

et al. 2010

Clin Vaccine Immunol

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In felids, three hemotropic mycoplasma species (hemoplasmas) have been described: Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum," and "Candidatus Mycoplasma turicensis." In particular, M. haemofelis may cause severe, potentially life-threatening hemolytic anemia. No routine serological assays for feline hemoplasma infections are available. Thus, the goal of our project was to identify and characterize an M. haemofelis antigen (DnaK) that subsequently could be applied as a recombinant antigen in a serological assay. The gene sequence of this protein was determined using consensus primers and blood samples from two naturally M. haemofelis-infected Swiss pet cats, an experimentally M. haemofelis-infected specific-pathogen-free cat, and a naturally M. haemofelis-infected Iberian lynx (Lynx pardinus). The M. haemofelis DnaK gene sequence showed the highest identity to an analogous protein of a porcine hemoplasma (72%). M. haemofelis DnaK was expressed recombinantly in an Escherichia coli DnaK knockout strain and purified using Ni affinity, sizeexclusion, and anion-exchange chromatography. It then was biochemically and functionally characterized and showed characteristics typical for DnaKs (secondary structure profile, thermal denaturation, ATPase activity, and DnaK complementation). Moreover, its immunogenicity was assessed using serum samples from experimentally hemoplasma-infected cats. In Western blotting or enzyme-linked immunosorbent assays, it was recognized by sera from cats infected with M. haemofelis, "Ca. Mycoplasma haemominutum," and "Ca. Mycoplasma turicensis," respectively, but not from uninfected cats. This is the first description of a full-length purified recombinant feline hemoplasma antigen that can readily be applied in future pathogenesis studies and may have potential for application in a diagnostic serological test.

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Section: Discussionsupporting

confidence: 76%

Identification, Characterization, and Application of a Recombinant Antigen for the Serological Investigation of Feline Hemotropic Mycoplasma Infections

Wolf-Jäckel

Jäckel

Museux

et al. 2010

Clin Vaccine Immunol

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“…Because the gram‐negative bacterium E. coli has been primarily used for previous investigations of DnaK and its co‐chaperones, therefore relatively few studies using gram‐positive bacterial DnaK chaperone systems have been reported . For example, Bacillus subtilis dnaK operon mutants can grow within a temperature range of 16°C–52°C and fail to form colonies above 52°C, in contrast to E. coli dnaK null mutants .…”

Section: Discussionmentioning

confidence: 99%

“…Only a few DnaK chaperone systems from gram‐positive bacteria have been investigated with respect to this property. However, it has been observed that the activity of the DnaK chaperone system in gram‐positive bacteria appears to vary with the species . The Clostridium acetobutylicum DnaK chaperone system is able to refold chemically denatured luciferase, albeit with a lower yield than the E. coli DnaK chaperone system .…”

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confidence: 99%

Recognizability of heterologous co‐chaperones with Streptococcus intermedius DnaK and Escherichia coli DnaK

Tomoyasu

Tsuruno

Tanatsugu

et al. 2018

Microbiology and Immunology

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Streptococcus intermediusDnaK complements the temperature-sensitive phenotype of an Escherichia coli dnaK null mutant only when co-chaperones DnaJ and GrpE are co-expressed. Therefore, whether S. intermedius DnaK and E. coli DnaK can recognize heterologous co-chaperones in vitro was examined. Addition of heterologous GrpE to DnaK and DnaJ partially stimulated adenosine triphosphatase (ATPase) activity, and almost completely stimulated luciferase refolding activity. Addition of heterologous DnaJ to GrpE and DnaK also stimulated ATPase activity; however, significant luciferase refolding activity was not observed. Moreover, E. coli DnaJ had a negative effect on the luciferase refolding activity of the S. intermedius DnaK chaperone system. In E. coli chaperone mutants, with the exception of E. coli DnaJ, stronger expression of the heterologous co-chaperones partially or almost completely complemented the temperature-sensitive-phenotype. These results indicate that all heterologous co-chaperones can at least partially recognize DnaK of a distantly related species. A region of the ATPase domain that is present in the DnaK of gram-negative bacteria is absent from the DnaK of gram-positive bacteria. This region is believed to be important for recognition of co-chaperones from gram-negative bacteria. However, insertion of this segment into S. intermedius DnaK failed to increase its ability to recognize E. coli co-chaperones, implying that this region is unnecessary or insufficient for the recognition of E. coli co-chaperones. Thus, our data suggest that a basic structural similarity is conserved among the components of the S. intermedius and E. coli DnaK chaperone systems, allowing weak associations between heterologous components.

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“…The role of dnak in thermoregulation is well established by gene expression studies at mRNA level ( Wetzstein et al, 1992 ) and deletion mutation studies ( Singh et al, 2007 ). Enhancement of thermotolerance in the heterologous system has been reported in various organisms like Escherichia coli ( Liang et al, 2009 ), tobacco ( Ono et al, 2001 ), Arabidopsis thaliana ( Montero-Barrientos et al, 2010 ) and Rice ( Uchida et al, 2008 ). dnaK gene from Trichoderma harzianum has been found to enhance tolerance to drought and freezing stress in poplar ( Takabe et al, 2008 ).…”

Section: Introductionmentioning

confidence: 98%

“…Five major families of Hsps are recognized; Hsp 70 (DnaK) family; the chaperonins (GroEL and Hsp 60); Hsp 90 family; Hsp 100 (Clp) family and the small Hsp (sHsp) family ( Wang et al, 2004 ). DnaK proteins are involved in de novo protein folding, membrane translocation, formation and disassembly of protein complexes and degradation of misfolded proteins ( Liang et al, 2009 ). DnaK consists of a highly conserved NH 2 -terminal ATPase domain, COOH-terminal substrate binding domain and a α-helical domain.…”

Section: Introductionmentioning

confidence: 99%

Cloning and expression of dnaK gene from Bacillus pumilus of hot water spring origin

Kumar

Prasanna

Lone

et al. 2014

Applied & Translational Genomics

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A set of thermotolerant strains isolated from hot springs of Manikaran and Bakreshwar (India) were selected with an aim to isolate dnak gene which encodes DnaK protein. The gene dnaK along with its flanking region was successfully amplified from 5 different strains (4 from Bakreshwar and one from Manikaran). Restriction fragment length polymorphism (RFLP) revealed that amplicons were almost identical in sequence. The dnak gene from one representative, Bacillus pumilus strain B3 isolated from Bakreshwar hot springs was successfully cloned and sequenced. The dnaK gene was flanked by gene grpE on one side. The dnaK gene was 1842 bp in length encoding a polypeptide of 613 amino acid residues. Calculated molecular weight and pI of the protein were 66,128.36 Da and 4.72 respectively. The deduced amino acid sequence of this gene shared high sequence homology with other DnaK proteins and its homologue Hsp 70 from other microorganisms, but possessed 36 substitutions and two insertions, as compared to DnaK protein of Bacillus subtilis. The dnaK gene of B. pumilus was successfully expressed in Escherichia coli BL 21 (DE3) using pET expression systems. Heterologous expression of dnaK of B. pumilus in E. coli BL 21 (DE3) allowed for the growth of E. coli up to 50 °C and survival up to 60 °C for 16 h, suggesting that dnak from B. pumilus imparts tolerance to host cells under high temperature. This novel gene can be an important component for possible utilization in abiotic stress management of plants.

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

Cited by 6 publications

References 53 publications

Identification, Characterization, and Application of a Recombinant Antigen for the Serological Investigation of Feline Hemotropic Mycoplasma Infections

Identification, Characterization, and Application of a Recombinant Antigen for the Serological Investigation of Feline Hemotropic Mycoplasma Infections

Recognizability of heterologous co‐chaperones with Streptococcus intermedius DnaK and Escherichia coli DnaK

Cloning and expression of dnaK gene from Bacillus pumilus of hot water spring origin

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