Recombinant Mesoplasma florum Chaperone protein DnaK (dnaK), partial

What is Mesoplasma florum and why is it significant as a model organism?

Mesoplasma florum is a bacterium with a near-minimal genome (approximately 800 kb) that has emerged as an important model organism for systems and synthetic biology. It was first isolated and described almost 40 years ago but remained relatively uncharacterized until recently. Following the publication of its first genome in 2004, M. florum has gained significance due to several key attributes:

• Small genome size (~800 kb) with approximately 651-740 protein-coding genes across different strains

• Fast growth rate compared to other minimal organisms

• Non-pathogenic nature (not associated with any disease)

• Potential for genome engineering and synthetic biology applications

These characteristics make M. florum an attractive model for understanding the minimal genetic requirements for cellular life and for developing simplified cellular chassis for synthetic biology applications .

What is the DnaK chaperone protein and what role does it play in Mesoplasma florum?

DnaK is a highly conserved molecular chaperone protein belonging to the heat shock protein 70 (Hsp70) family. In M. florum, as in other bacteria, DnaK likely plays crucial roles in:

• Protein folding and quality control

• Prevention of protein aggregation during cellular stress

• Assisting in protein transport across membranes

• Protection of cells against various stress conditions (heat shock, oxidative stress)

• Potential involvement in DNA replication and repair processes

Given M. florum's minimal genome, understanding the functions of conserved proteins like DnaK provides insight into essential cellular processes that have been retained through reductive evolution.

How does M. florum DnaK compare to DnaK proteins in other bacterial species?

While specific comparative data for M. florum DnaK is limited in the provided search results, we can infer that:

• M. florum DnaK likely retains the core functional domains (nucleotide-binding domain and substrate-binding domain) found in other bacterial DnaK proteins

• As part of a minimal organism, M. florum DnaK may have undergone streamlining while preserving essential functions

• Comparative genomics studies have shown that approximately 80% of all protein-coding genes are conserved across M. florum strains, with translation-related functions being significantly enriched in this core genome

Researchers should perform sequence analysis and structural predictions to better characterize these relationships when working with recombinant M. florum DnaK.

What are the optimal expression systems for recombinant M. florum DnaK production?

When expressing recombinant M. florum DnaK, researchers should consider:

Escherichia coli expression systems:

• BL21(DE3) strains are recommended for high-level expression

• Consider using chaperone co-expression systems (such as GroEL/GroES) to enhance proper folding

• Codon optimization may be necessary due to the different codon usage between M. florum and E. coli

Expression conditions optimization:

• Induction at lower temperatures (16-25°C) often improves solubility

• IPTG concentration should be titrated (typically 0.1-1.0 mM)

• Expression time should be optimized (4-16 hours)

Alternative expression systems such as cell-free protein synthesis might also be considered for difficult-to-express variants.

What purification strategies yield the highest purity and activity of recombinant M. florum DnaK?

A multi-step purification approach is recommended:

• Affinity chromatography:

  • His-tagged DnaK can be purified using Ni-NTA resins

  • GST-tagged versions can utilize glutathione sepharose

• Ion exchange chromatography:

  • Usually using Q-sepharose at pH 8.0

• Size exclusion chromatography:

  • Final polishing step to remove aggregates and ensure homogeneity

Buffer optimization:

• Presence of nucleotides (ATP/ADP) may stabilize the protein

• Addition of low concentrations of reducing agents (1-5 mM DTT or β-mercaptoethanol)

• Glycerol (5-10%) often improves stability during storage

Protein purity should be assessed via SDS-PAGE and activity via ATPase assays.

How can researchers verify the functionality of recombinant M. florum DnaK?

Functional verification should include multiple complementary approaches:

Biochemical assays:

• ATPase activity measurement (colorimetric or coupled enzymatic assays)

• Substrate binding assays using model peptides (fluorescence anisotropy)

• Protein aggregation prevention assays (using model substrates like citrate synthase)

Structural confirmation:

• Circular dichroism to verify secondary structure

• Limited proteolysis to confirm correct folding

• Thermal shift assays to assess protein stability

Functional complementation:

• Rescue of E. coli dnaK mutants under stress conditions

• Chaperone activity assays in vitro

Researchers should also verify the nucleotide-binding capacity of the recombinant protein, as this is essential for DnaK function.

How can recombinant M. florum DnaK be used in synthetic biology applications?

Recombinant M. florum DnaK has several potential applications in synthetic biology:

Minimal cell design:

• Understanding the role of DnaK in M. florum can inform the design of minimal synthetic cells

• The JCVI-syn3.0 project demonstrated that molecular chaperones are essential for minimal cells, suggesting DnaK likely plays a crucial role in M. florum viability

Protein folding enhancement:

• Co-expression of M. florum DnaK could potentially enhance the production of difficult-to-express proteins

• Particularly valuable if M. florum DnaK has unique substrate specificity or stability properties

Stress resistance engineering:

• Insights from M. florum DnaK could be applied to engineer stress-resistant strains for biotechnology applications

Biotechnological applications:

• Development of protein folding biosensors

• Creation of heat-stable enzyme production systems

What techniques are available for studying DnaK interactions with other components of the M. florum proteostasis network?

Several approaches can be employed:

Co-immunoprecipitation and pull-down assays:

• Using tagged recombinant DnaK to identify interaction partners

• Followed by mass spectrometry for protein identification

Crosslinking mass spectrometry:

• Captures transient interactions within the chaperone network

• Provides structural information about interaction interfaces

Proximity labeling:

• BioID or APEX2 fusions to DnaK to identify proximal proteins in vivo

• Particularly useful for capturing weak or transient interactions

Fluorescence-based approaches:

• FRET assays to study DnaK-substrate interactions

• Fluorescence recovery after photobleaching (FRAP) to study dynamics

Genetic approaches:

• Synthetic genetic array analysis to identify genetic interactions

• Suppressor screens to identify functional relationships

These approaches would be particularly valuable in the context of M. florum's minimal genome, as they could reveal essential chaperone-substrate relationships that have been conserved during genome reduction.

How can genome engineering tools for M. florum be used to study DnaK function in vivo?

Recent developments in M. florum genetic tools enable several approaches:

Plasmid-based expression:

• The oriC-based plasmids developed for M. florum can be used to express modified versions of DnaK

• Different versions of these plasmids have transformation frequencies of approximately 4.1 × 10⁻⁶ transformants per viable cell

Genome editing possibilities:

• Whole genome cloning (WGC) of M. florum in yeast provides a platform for genome engineering

• Recombineering using λ-Red system or GP35 recombinase could be adapted from other Mollicutes

• CRISPR-Cas9 technology could potentially be adapted for M. florum genome editing

Transformation methods:

• Polyethylene glycol (PEG)-mediated transformation

• Electroporation (reaching frequencies up to 7.87 × 10⁻⁶ transformants per viable cell)

• Conjugation from E. coli (reaching frequencies up to 8.44 × 10⁻⁷ transformants per viable cell)

Selection markers:

• Tetracycline resistance (tetM gene)

• Puromycin resistance

• Spectinomycin/streptomycin resistance

These tools could enable precise manipulation of dnaK in its native context to study its physiological functions.

What are the common challenges in working with recombinant M. florum DnaK and how can they be addressed?

Researchers may encounter several challenges:

Solubility issues:

• Problem: Recombinant DnaK may form inclusion bodies

• Solution: Lower induction temperature (16-18°C), use solubility tags (SUMO, MBP), or co-express with chaperones

Activity loss during purification:

• Problem: DnaK may lose ATPase activity or substrate binding capacity

• Solution: Include ATP/ADP in purification buffers, avoid multiple freeze-thaw cycles, optimize buffer conditions

Co-purifying contaminants:

• Problem: Endogenous E. coli chaperones or substrates may co-purify

• Solution: Include high-salt washes, ATP-dependent washing steps, and multiple chromatography steps

Oligomerization:

• Problem: DnaK may form dimers or higher-order oligomers

• Solution: Include reducing agents, optimize protein concentration, and perform final size-exclusion chromatography

Post-translational modifications:

• Problem: M. florum may have specific modifications not present in heterologous systems

• Solution: Consider native purification from M. florum itself using the newly developed genetic tools

How can researchers integrate DnaK studies with M. florum systems biology approaches?

Integration of DnaK studies with systems biology requires multi-omics approaches:

Transcriptomics integration:

• RNA-seq under various stress conditions to understand dnaK regulation

• Correlation of DnaK expression with other genes in the stress response network

Proteomics approaches:

• Global proteome analysis to identify DnaK substrates

• Thermal proteome profiling to identify proteins stabilized by DnaK in vivo

• Quantitative proteomics to measure protein abundance changes in DnaK mutants

Metabolomics correlation:

• Connecting changes in metabolite profiles with DnaK activity

• Identifying metabolic pathways affected by DnaK function

Network modeling:

• Integration of DnaK into genome-scale metabolic models

• Development of mathematical models of the M. florum chaperone network

The high-quality genome-scale metabolic model already developed for M. florum provides an excellent foundation for integrating DnaK function into systems-level analyses .

What considerations are important when comparing results from recombinant DnaK versus native M. florum DnaK?

Researchers should consider several factors:

Expression system differences:

• Codon usage optimization may alter protein folding kinetics

• Post-translational modifications may differ between native and recombinant systems

• Expression level differences may affect activity measurements

Buffer and environmental conditions:

• pH, salt concentration, and crowding agents should mimic the M. florum cytoplasmic environment

• Temperature ranges should reflect M. florum's natural growth conditions

Co-factor availability:

• Ensure appropriate co-chaperones (DnaJ, GrpE) are available for functional studies

• Consider the presence of specific nucleotide exchange factors

Structural integrity validation:

• Compare circular dichroism spectra between native and recombinant proteins

• Perform limited proteolysis to confirm similar folding patterns

• Validate by comparing thermal stability profiles

Functional benchmarking:

• Compare ATPase activity rates between native and recombinant proteins

• Validate substrate specificity using identical model substrates

How might M. florum DnaK research contribute to understanding minimal cellular requirements?

Research on M. florum DnaK could provide several insights into minimal cellular requirements:

Essential chaperone functions:

• Identifying which specific functions of DnaK are indispensable in a near-minimal organism

• Determining the minimal substrate repertoire that must be maintained for viability

Co-evolution with minimal proteome:

• Understanding how DnaK has co-evolved with the reduced proteome of M. florum

• Identifying specialized adaptations that enable efficient chaperoning with limited resources

Comparison with synthetic minimal cells:

• Comparing DnaK essentiality in M. florum with that in JCVI-syn3.0

• Determining if alternative protein quality control mechanisms exist in minimal cells

Stress response in minimal organisms:

• Characterizing how stress response pathways function with minimal genetic components

• Understanding trade-offs between genome minimization and stress resilience

The core genome of M. florum contains approximately 546 homologous gene cluster families, representing about 80% of all protein-coding genes in each strain . Understanding DnaK's role within this core set of genes would provide valuable insights into the essential functions that must be maintained in minimal cells.

What novel approaches could be developed to study DnaK-substrate interactions specific to M. florum?

Several innovative approaches could be developed:

M. florum-specific peptide arrays:

• Design of peptide arrays representing the M. florum proteome

• Screening for specific binding motifs preferred by M. florum DnaK

In vivo proximity labeling adapted to M. florum:

• Development of BioID or TurboID systems functional in M. florum

• Identification of native substrates in their cellular context

Single-molecule studies:

• FRET-based approaches to study DnaK-substrate binding dynamics

• Single-molecule force spectroscopy to study the mechanics of substrate unfolding

Cryo-EM structural studies:

• Visualization of DnaK-substrate complexes at high resolution

• Comparison with DnaK structures from other organisms

Synthetic biology approaches:

• Creation of minimal DnaK variants with reduced functionality

• Testing which domains and functions are essential in the M. florum context

These approaches would benefit from the genetic tools recently developed for M. florum, including oriC-based plasmids that enable genetic manipulation .

How might insights from M. florum DnaK contribute to the development of synthetic minimal cells?

M. florum DnaK research could contribute to synthetic minimal cells in several ways:

Design principles for minimal chaperone networks:

• Defining the minimal set of chaperone interactions needed for cellular viability

• Identifying essential substrate categories that require chaperoning

Stress tolerance engineering:

• Understanding how DnaK contributes to stress tolerance in minimal genomes

• Engineering optimized DnaK variants for synthetic cells with enhanced stress resistance

Protein folding efficiency:

• Identifying sequence determinants that enhance folding efficiency in minimal proteomes

• Designing proteins with reduced chaperoning requirements

Integration with whole-cell models:

Genome reduction strategies:

• Informing future genome reduction efforts by highlighting essential chaperone functions

• Guiding the retention or modification of DnaK-dependent pathways in minimal genomes

The successful whole genome cloning of M. florum in yeast provides an excellent platform for testing synthetic biology hypotheses related to DnaK function and for potentially creating further reduced genomes based on insights gained from DnaK studies.