Recombinant Mesoplasma florum Deoxyribose-phosphate aldolase 1 (deoC1)

What is Recombinant Mesoplasma florum Deoxyribose-phosphate aldolase 1 (deoC1)?

Recombinant Mesoplasma florum Deoxyribose-phosphate aldolase 1 (deoC1) is a purified enzyme encoded by the deoC1 gene from the near-minimal bacterium Mesoplasma florum. According to the Uniprot database (entry Q6F1Z6), this enzyme is classified as EC 4.1.2.4 and is also known as DERA 1, 2-deoxy-D-ribose 5-phosphate aldolase 1, phosphodeoxyriboaldolase 1, or deoxyriboaldolase 1 . The recombinant protein is expressed from the full-length sequence (residues 1-220) and is typically produced with a purity exceeding 85% as determined by SDS-PAGE analysis . The protein sequence begins with MKLNKYIDHT and continues through the functional domains characteristic of aldolase enzymes involved in nucleoside metabolism .

What is the enzymatic function of deoC1?

Deoxyribose-phosphate aldolase 1 (deoC1) catalyzes the reversible aldol reaction between acetaldehyde and D-glyceraldehyde 3-phosphate to form 2-deoxy-D-ribose 5-phosphate. This reaction represents a critical step in the catabolism of deoxyribonucleosides and nucleotides, which is essential for recycling nucleic acid components. In the catabolic direction, deoC1 cleaves 2-deoxy-D-ribose 5-phosphate into acetaldehyde and D-glyceraldehyde 3-phosphate. The enzyme can also catalyze aldol additions with various aldehyde acceptors, making it potentially useful for stereoselective carbon-carbon bond formation in synthetic applications. In the context of Mesoplasma florum, which has a minimal genome, this enzyme plays a crucial role in optimized nucleoside metabolism pathways.

Why is studying deoC1 from Mesoplasma florum significant for research?

Studying deoC1 from Mesoplasma florum is significant for several compelling reasons. First, M. florum is considered a near-minimal bacterium, making it an attractive model for systems biology and synthetic biology research . As noted in recent studies, M. florum represents a potential simplified cell chassis for synthetic biology applications, and understanding the function of its essential enzymes like deoC1 provides insight into minimal requirements for cellular metabolism . Second, aldolases are important biocatalysts with potential applications in stereoselective synthesis of complex carbohydrates and other compounds. Third, comparative studies of deoC1 across species can reveal evolutionary adaptations in nucleoside metabolism pathways. Investigating M. florum's deoC1 contributes to our understanding of core metabolic functions in minimal cellular systems that have evolved for maximum efficiency with minimal genetic complexity.

What are the optimal storage conditions for recombinant deoC1?

According to product documentation, the optimal storage conditions for recombinant Mesoplasma florum deoC1 depend on its formulation. For liquid formulations, the recommended storage is at -20°C to -80°C, with an expected shelf life of approximately 6 months under these conditions . For lyophilized (freeze-dried) preparations, storage at -20°C to -80°C extends the shelf life to approximately 12 months . The actual stability is influenced by multiple factors beyond temperature, including buffer composition and the intrinsic stability of the protein itself . To maximize stability and functionality, researchers should:

• Aliquot the protein upon initial thawing to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for no more than one week

• Use appropriate stabilizing agents such as glycerol (recommended at 5-50% final concentration) for long-term storage

• Ensure sterile conditions when handling the protein

• Monitor activity periodically using appropriate enzyme assays

Repeated freezing and thawing is explicitly not recommended as it can lead to protein denaturation and loss of enzymatic activity .

How should recombinant deoC1 be reconstituted for laboratory use?

Proper reconstitution of recombinant Mesoplasma florum deoC1 is critical for maintaining enzymatic activity and stability in laboratory applications. Based on product documentation, the following methodological protocol is recommended:

• Initial preparation:

  • Centrifuge the vial briefly before opening to bring the contents to the bottom of the tube

  • Allow the sealed vial to equilibrate to room temperature if removed from freezer storage

  • Work in a clean environment to minimize contamination

• Reconstitution procedure:

  • Use deionized, sterile water as the primary reconstitution buffer

  • Add water to achieve a protein concentration between 0.1-1.0 mg/mL

  • Add the water gently to the sides of the vial rather than directly onto the protein

  • Allow the protein to dissolve completely without aggressive mixing

• Stabilization:

  • Add glycerol to a final concentration of 5-50% as recommended for long-term stability

  • Consider adding reducing agents if the protein contains cysteine residues susceptible to oxidation

  • Adjust the pH if necessary to maintain optimal conditions (typically pH 7.0-7.5 for aldolases)

• Aliquoting and storage:

  • Divide the reconstituted protein into small, single-use aliquots

  • Store working aliquots at 4°C for up to one week

  • For longer storage, keep aliquots at -20°C/-80°C with added glycerol (5-50%)

Following these methodological steps ensures maximum retention of enzymatic activity and extends the usable lifetime of the recombinant deoC1 preparation.

What expression systems are used for producing recombinant deoC1?

The production of functional recombinant Mesoplasma florum deoC1 requires careful selection of expression systems to ensure proper folding and activity. Based on available information and general practices in recombinant protein production, the following expression systems and methodological considerations are relevant:

• Yeast expression systems:

  • Product documentation indicates that yeast has been successfully used as a host for M. florum deoC1 expression

  • Advantages include eukaryotic folding machinery and secretion capacity

  • Common yeast platforms include Saccharomyces cerevisiae and Pichia pastoris

  • Methodological considerations include codon optimization, signal sequence selection, and induction condition optimization

• Bacterial expression systems:

  • While not explicitly mentioned for M. florum deoC1, E. coli is commonly used for aldolase expression

  • Strains designed for enhanced disulfide bond formation may improve folding

  • Temperature optimization (often lower temperatures of 16-25°C) can improve solubility

• Tag selection and design considerations:

  • N-terminal vs. C-terminal tag placement can significantly impact folding and activity

  • For M. florum deoC1, product documentation indicates that "tag type will be determined during the manufacturing process," suggesting optimization is required

• Expression verification:

  • SDS-PAGE analysis (product documentation indicates >85% purity is achievable)

  • Activity assays to confirm functional expression

The selection of an appropriate expression system should be guided by the intended application, required yield, and importance of specific post-translational modifications for enzymatic activity.

What challenges occur in expressing and purifying functional recombinant deoC1?

Researchers working with recombinant Mesoplasma florum deoC1 encounter several challenges during expression and purification that require specific methodological approaches:

• Expression system selection challenges:

  • M. florum has different codon usage patterns and lacks certain post-translational modification systems

  • Solutions include:

    • Optimize codon usage for the expression host

    • Test multiple expression strains and conditions

    • Consider fusion tags that enhance solubility

    • Evaluate lower induction temperatures (16-20°C) to promote proper folding

• Maintaining enzymatic activity:

  • Deoxyribose-phosphate aldolase activity is sensitive to oxidation and certain buffer conditions

  • Strategies include:

    • Adding reducing agents to all buffers

    • Using oxygen-depleted buffers when possible

    • Including glycerol (10-20%) to stabilize protein structure

    • Determining optimal pH range for storage buffers

• Purification challenges:

  • Selective purification while maintaining activity requires:

    • IMAC (immobilized metal affinity chromatography) with appropriate tags

    • Size exclusion chromatography to ensure homogeneity

    • Activity assays at each purification step to monitor functional protein recovery

• Storage stability limitations:

  • As indicated in the product documentation, recombinant deoC1 has limited shelf life

  • Strategies to extend usability include:

    • Lyophilization in the presence of appropriate excipients

    • Adding stabilizers like trehalose or sucrose

    • Aliquoting to avoid freeze-thaw cycles

• Activity verification:

  • Developing appropriate assays such as:

    • Spectrophotometric assays coupling acetaldehyde production to NADH oxidation

    • Direct measurement of substrate consumption by HPLC

    • Coupled enzyme assays to monitor product formation

These methodological considerations are essential for obtaining functionally active deoC1 suitable for enzymatic studies and biotechnological applications.

How does deoC1 from M. florum compare to homologs from other bacterial species?

A comparative analysis of deoC1 from Mesoplasma florum with homologs from other bacterial species reveals both conserved features and unique characteristics. The M. florum deoC1 (UniProt: Q6F1Z6) consists of 220 amino acids and shares the core catalytic mechanism with other deoxyribose-phosphate aldolases . Below is a comparative analysis of key features:

*Estimated values based on typical properties of related enzymes and organisms

Structural analysis suggests that while the catalytic core is conserved across species, M. florum deoC1 exhibits unique surface charges and loop regions that may reflect adaptation to its minimal cellular environment. The enzyme from M. florum likely evolved for maximal efficiency with minimal structural complexity, consistent with the organism's reduced genome .

Phylogenetic studies indicate that deoC1 from Mollicutes (including M. florum) forms a distinct clade compared to enzymes from other bacterial phyla, reflecting their divergent evolution. These comparative differences make M. florum deoC1 particularly interesting for both basic research on enzyme evolution and potential applications in synthetic biology where minimal, efficient enzymes are desirable.

How can oriC-based plasmids be developed for studying deoC1 function in M. florum?

Developing oriC-based plasmids for studying deoC1 function in M. florum requires careful consideration of the replication origins and genetic elements. Based on recent research, successful plasmid development should follow these methodological steps:

• Include both the rpmH-dnaA and dnaA-dnaN intergenic regions in the plasmid design. Research has demonstrated that plasmids harboring both these regions result in transformation frequencies of approximately 4.1 × 10^-6 transformants per viable cell, while plasmids containing only one region fail to produce detectable transformants .

• Consider including a copy of the dnaA gene between these regions, although research shows that plasmids both with and without dnaA can be stably maintained throughout multiple generations .

• Select appropriate antibiotic resistance markers. Functional resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin have been demonstrated to work effectively in M. florum .

• Account for recombination tendencies. Southern blotting analysis reveals that oriC plasmids have a strong tendency to recombine with the chromosomal oriC region, regardless of whether they contain a copy of the dnaA gene . This recombination typically occurs at the dnaA-dnaN intergenic region .

• For transformation, multiple methods have proven effective with different efficiencies (discussed in section 5.2).

• For functional studies of deoC1, design the plasmid to either overexpress the native deoC1, express a modified version, or create a complementation system for studying the effects of deoC1 mutations or deletions.

It's worth noting that heterologous oriC regions from related species like Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri have been tested but failed to produce detectable transformants in M. florum .

What transformation methods are effective for introducing genetic constructs into M. florum?

Transformation of Mesoplasma florum with genetic constructs requires specialized methods due to the unique characteristics of this near-minimal bacterium. Recent research has validated three effective transformation approaches, each with distinct methodological considerations:

• Polyethylene Glycol (PEG)-mediated transformation:

  • Transformation frequency: Approximately 4.1 × 10^-6 transformants per viable cell

  • Methodology: Involves treating M. florum cells with a buffer containing PEG, which makes the cell membrane temporarily permeable to DNA

  • Advantages: Well-established protocol with consistent results

  • Limitations: May cause cell toxicity at higher PEG concentrations

• Electroporation:

  • Transformation frequency: Up to 7.87 × 10^-6 transformants per viable cell (highest efficiency among tested methods)

  • Methodology: Application of high-voltage electrical pulses that create temporary pores in the cell membrane

  • Critical parameters: Field strength, pulse duration, cell density, DNA concentration

  • Advantages: Higher efficiency than PEG-mediated transformation; faster procedure

• Conjugation from Escherichia coli:

  • Transformation frequency: Up to 8.44 × 10^-7 transformants per viable cell

  • Methodology: DNA transfer through direct cell-to-cell contact between a donor E. coli strain and recipient M. florum cells

  • Advantages: Does not require preparation of competent cells; can transfer larger DNA constructs

  • Limitations: Lower efficiency; requires establishing optimal donor:recipient ratios

For all methods, successful transformation is highly dependent on the design of the genetic construct, particularly the replication origin. Selection of transformed cells typically employs antibiotic resistance markers, with demonstrated success using genes conferring resistance to tetracycline, puromycin, and spectinomycin/streptomycin in M. florum .

How can the stability of deoC1 be maintained during experimental procedures?

Maintaining the stability and activity of deoC1 during experimental procedures requires careful attention to multiple factors that impact protein integrity. Based on both general protein handling principles and specific information about deoxyribose-phosphate aldolases, researchers should implement the following methodological strategies:

• Temperature management:

  • Keep the enzyme at 4°C during all purification and handling steps

  • Avoid room temperature exposure for extended periods

  • For longer procedures, use ice baths or refrigerated chambers

  • Never rapidly heat or cool the protein solution, as this can cause denaturation

• Buffer optimization:

  • Maintain pH in the optimal range (typically 7.0-7.5 for most aldolases)

  • Include stabilizing agents such as glycerol (10-20%)

  • Add reducing agents to prevent oxidation of cysteine residues

  • Consider adding metal cofactors if required for structural stability

• Storage formulation:

  • For short-term storage (up to 1 week), maintain at 4°C in appropriate buffer

  • For long-term storage, add glycerol to 5-50% final concentration and store at -20°C/-80°C

  • Prepare single-use aliquots to avoid freeze-thaw cycles

  • Consider lyophilization with appropriate excipients for extended shelf life

• Protection against proteolysis:

  • Add protease inhibitors during extraction and purification steps

  • Remove cellular proteases early in the purification process

  • Minimize handling time to reduce exposure to potential proteases

• Activity preservation:

  • Periodically verify enzymatic activity using appropriate assays

  • Store with substrate analogs or competitive inhibitors to stabilize active site conformation

  • Document activity decay rates under different storage conditions

These methodological approaches should be systematically tested and optimized for the specific properties of M. florum deoC1 to establish a robust protocol for maintaining enzyme stability and activity.

What strategies can overcome recombination issues when using oriC-based plasmids in M. florum?

Working with oriC-based plasmids in Mesoplasma florum presents significant challenges due to their tendency to recombine with the chromosomal oriC region. Southern blotting analysis has shown that all tested clones exhibited recombination events with the host chromosome, with the majority occurring at the dnaA-dnaN intergenic region . To address these recombination issues, researchers can implement several strategic approaches:

• Sequence modifications to reduce homologous recombination:

  • Introduce silent mutations in the plasmid oriC sequences to reduce sequence identity while maintaining functionality

  • Design minimal oriC regions containing only essential elements for replication

  • Use heterologous sequences from closely related species that maintain functionality but have reduced homology

• Plasmid design considerations:

  • Incorporate genetic elements that provide selection against chromosomal integration

  • Include counter-selectable markers that become detrimental when integrated

  • Design plasmids with increased copy number to favor extrachromosomal maintenance

• Experimental protocols to enrich for extrachromosomal forms:

  • Optimize transformation procedures to favor maintenance of extrachromosomal elements

  • Develop screening methods to identify clones with predominantly extrachromosomal plasmids

  • Implement growth conditions that select against recombinants

• Monitoring and quantification strategies:

  • Regularly assess plasmid status using Southern blotting or PCR-based methods

  • Quantify extrachromosomal versus integrated forms using qPCR

  • Track plasmid stability over multiple generations under different selective pressures

It's worth noting that recent research has shown that up to 71% of analyzed clones (17 out of 24) maintained the oriC plasmids as extrachromosomal elements, though all of these also showed recombination events with the oriC region of the M. florum chromosome . This suggests that a heterogeneous population of cells with different plasmid states emerges from a single initial colony, which researchers must account for in experimental design and data interpretation.

How might deoC1 contribute to synthetic biology applications using M. florum?

Mesoplasma florum is considered a promising chassis for synthetic biology due to its near-minimal genome, and deoC1 plays a potentially significant role in these applications . The enzyme's involvement in nucleoside metabolism makes it relevant for several synthetic biology approaches:

• Minimal genome projects:

  • Understanding whether deoC1 is essential in a minimal cell design

  • Determining the minimum functional domains required for activity

  • Engineering optimized versions with enhanced stability or catalytic efficiency

• Metabolic pathway engineering:

  • Incorporation into synthetic pathways for nucleotide salvage

  • Modification for expanded substrate specificity to enable novel reactions

  • Integration with other enzymes for complete synthetic pathways

• Biocontainment strategies:

  • Engineering deoC1 dependency on non-natural substrates

  • Creating synthetic auxotrophy systems that require specific deoC1 activity

  • Developing kill switches based on controlled deoC1 expression

• Biocatalysis applications:

  • Utilizing deoC1's aldol addition capacity for stereoselective synthesis

  • Engineering substrate specificity for production of valuable compounds

  • Immobilization strategies for industrial biocatalysis

The successful development of genetic tools for M. florum, particularly the oriC-based plasmids and transformation methods, provides the foundation for these applications by enabling genetic manipulation of deoC1 and other genes in this minimal organism .