Recombinant Chaperone protein DnaK (dnaK)

What is DnaK and what is its primary function in bacterial cells?

DnaK is a molecular chaperone belonging to the Hsp70 family in bacteria. In Escherichia coli, DnaK interacts with a wide range of newly synthesized polypeptides and assists their proper folding and assembly into oligomers by preventing protein aggregation . Its primary functions include:

• Assisting in the folding of newly synthesized proteins

• Preventing aggregation of partially folded proteins

• Disaggregating preformed protein aggregates (in cooperation with ClpB ATPase)

• Participating in the degradation of damaged proteins by Lon and ClpP proteases

Research approaches to investigate these functions typically involve genetic manipulation of dnaK expression, protein-protein interaction studies, and in vitro folding assays with model substrate proteins.

How does DnaK essentiality differ across bacterial species?

The requirement for DnaK varies significantly between bacterial species, with important implications for experimental design:

In E. coli, deletion of dnaK leads to cell death under heat shock conditions, but at intermediate and low temperatures, ΔdnaK E. coli cells remain viable despite growth defects . This non-essentiality has been attributed to the high degree of functional redundancy in the chaperone network and the finding that few DnaK clients are essential proteins .

In contrast, C. crescentus strictly depends on DnaK at all temperature conditions due to its regulatory function in gene expression and DNA replication . In M. smegmatis, DnaK has a non-redundant role in the folding of essential proteins and in generally maintaining protein homeostasis .

Why is DnaK frequently a contaminant in recombinant protein purification?

DnaK contamination represents a significant technical challenge in protein purification because:

• DnaK's biological function involves binding to unfolded or partially folded proteins

• The ability of DnaK to bind to regions where the polypeptide chain is exposed makes its removal during purification challenging

• DnaK can co-purify with the target protein through multiple chromatographic steps

This contamination can interfere with downstream applications such as:

• Structural studies (crystallography, NMR, cryo-EM)

• Activity assays where DnaK might influence protein function

• Protein-protein interaction studies

Standard detection methods for DnaK contamination include SDS-PAGE (where DnaK appears at ~70 kDa), Western blotting with anti-DnaK antibodies, and mass spectrometry analysis.

What methodologies have been developed to remove DnaK contamination from purified recombinant proteins?

Researchers have developed several approaches to address DnaK contamination:

Genetic approach: DnaK-deficient expression strains

Using a BL21(DE3) ΔdnaK strain of Escherichia coli eliminates unavoidable contamination of purified recombinant proteins by DnaK . Studies have demonstrated that selected proteins expressed in this system remained soluble, correctly assembled, and active, establishing DnaK dispensability for protein production in the Lon protease-deficient BL21(DE3) strain .

Biochemical approach: GST-cleanser protein methodology

A novel method captures contaminating DnaK using a GST-tagged cleanser protein with functional binding sites for the chaperone . The procedure follows these steps:

• Co-incubate contaminated protein preparation with the GST-cleanser protein in the presence of ATP-Mg

• Allow the cleanser protein to capture DnaK through its binding sites

• Remove the GST-cleanser/DnaK complex using glutathione affinity chromatography

This approach substantially eliminates DnaK contamination from histidine-tagged recombinant proteins in a batch process and can be applied to proteins obtained with different expression and purification systems .

How does DnaK overexpression affect bacterial proteome evolution and mutational robustness?

Experimental evolution studies have revealed DnaK's role in mutational buffering:

Experimental design for studying DnaK's effect on protein evolution

Researchers conducted mutation accumulation experiments using:

• 68 parallel clonal lines derived from a hypermutable strain expressing DnaK under an inducible promoter (DnaK+)

• Evolution through repeated single-cell bottlenecks (creating strong genetic drift)

• 30 lines evolved at 37°C and 30 at 42°C (to increase the effect of destabilizing mutations)

• 16 control lines without DnaK overexpression capability (DnaK-)

• Evolution for approximately 1,870 generations

Key findings on mutational robustness

Genome sequencing of evolved lines revealed:

• DnaK overexpression maintained throughout the experiment despite potential energetic costs

• Increased robustness to nonsynonymous mutations in DnaK client proteins

• Strong DnaK clients showed higher tolerance to mutations than weak clients or non-clients

• This supports the hypothesis that proteins with greater dependence on DnaK have higher mutation tolerance

These findings demonstrate DnaK's role as a source of mutational robustness, with important implications for understanding protein evolution and designing directed evolution experiments.

What is the regulatory role of DnaK in gene expression and how does it contribute to essentiality in bacteria?

In Caulobacter crescentus, DnaK has essential regulatory functions beyond protein folding:

Transcriptional regulation mechanism

Research has demonstrated that in C. crescentus, DnaK's essential function under non-stress conditions is to inhibit the heat shock sigma factor σ32 . DnaK depletion leads to:

• Activation of σ32-dependent transcription

• Global reprogramming of gene expression

• Re-organization of cellular activities from proliferative to cytoprotective modes

Impact on DNA replication

DnaK depletion in C. crescentus also affects DNA replication by:

• Inducing degradation of the replication initiator DnaA through upregulation of the Lon protease

• Clearing DnaA from DnaK-depleted cells

• Inhibiting DNA replication initiation

Interestingly, depletion of DnaK does not lead to major protein aggregation in C. crescentus under non-stress conditions , suggesting its essential function is primarily regulatory rather than related to maintaining protein homeostasis.

How does the structure of DnaK-GrpE complexes inform our understanding of chaperone function?

Structural studies have provided insights into DnaK regulation by its nucleotide exchange factor GrpE:

• Two co-existing conformations of the DnaK-GrpE complex have been identified through advanced image processing techniques

• The first 33 GrpE residues act as a pseudo-substrate that competes with substrates for the DnaK binding site

• These structural details help explain the allosteric regulation of DnaK's ATPase cycle

The structural analysis employed sophisticated techniques:

• Initial processing assuming a unique conformation

• Generation of two reconstructions with reference-free averages

• Three-dimensional classification using maximum-likelihood methods

• Separate refinement of particles corresponding to each conformation

These insights provide a foundation for understanding the molecular mechanisms of DnaK regulation and for designing modulators of DnaK function.

What experimental considerations are important when using DnaK-deficient strains for protein expression?

When using DnaK-deficient strains for recombinant protein production, researchers should consider:

Impact on protein solubility and activity

• While proteins produced in BL21(DE3) ΔdnaK strains can remain soluble and active , DnaK deletion may increase insoluble fractions of aggregation-prone proteins

• Effects are protein-specific and require case-by-case evaluation

Alternative chaperone systems

In DnaK's absence:

• The GroEL/GroES system may compensate for some folding functions

• ClpB disaggregase effectiveness may be reduced, as it normally functions together with DnaK

Optimization strategies

• Temperature optimization: Lower expression temperatures to reduce aggregation

• Modified induction: Slower induction with lower inducer concentrations

• Co-expression of other chaperones if needed

• Additional quality control of the purified protein's folding state and activity

These considerations highlight the need for careful optimization when using DnaK-deficient strains, balancing reduced chaperone contamination against potential effects on protein folding.