Recombinant Debaryomyces hansenii Mitochondrial genome maintenance protein MGM101 (MGM101)

Methodological Questions

• What are the optimal conditions for expressing recombinant D. hansenii MGM101?

  Optimal expression of recombinant D. hansenii MGM101 requires careful consideration of several parameters:

  1. Expression system selection:

     • E. coli system: Most commonly used, offering high yield but potential folding issues

     • Yeast system: Provides more natural folding environment and post-translational modifications

     • Baculovirus system: Good for complex eukaryotic proteins requiring specific modifications

     • Mammalian cell system: For highest authenticity but typically lower yields

  2. Expression construct design:

     • Fusion partners like MBP significantly improve solubility and stability

     • Cleavage sites for releasing the native protein (e.g., Prescission™ protease site)

     • Codon optimization for the expression host

  3. Culture conditions:

     • Temperature: Often lower temperatures (16-20°C) improve folding

     • Induction parameters: IPTG concentration and induction timing for E. coli systems

     • Media composition: Rich media generally yields higher protein levels

  4. Extraction protocols:

     • Cell lysis buffer composition (typically containing protease inhibitors)

     • Sonication or mechanical disruption parameters

     • Centrifugation conditions to separate soluble and insoluble fractions

  A recommended protocol based on successful expression of yeast MGM101 includes expression as an MBP fusion in E. coli, culture at 18°C after induction, and extraction in a buffer containing 20mM Tris-HCl (pH 7.4), 200mM NaCl, 1mM EDTA, and protease inhibitors .

• What purification strategies yield the highest quality recombinant MGM101?

  A multi-step purification strategy is recommended for obtaining high-quality MGM101:

  1. Affinity chromatography:

     • MBP fusion allows purification using amylose resin

     • Incubate cell lysate with amylose resin at 4°C overnight

     • Wash thoroughly with column buffer

     • Elute with 10mM maltose in column buffer

  2. Proteolytic cleavage:

     • Cleave with Prescission™ protease (0.33 units/100 μg) overnight to release MGM101

     • The recombinant MGM101 will have additional amino acids (e.g., GPKVPEFGS) at the N-terminus

  3. Ion exchange chromatography:

     • Dialyze against 10mM sodium phosphate (pH 7.2, 4°C, 100mM NaCl)

     • Separate MGM101 from MBP using cation exchange chromatography

     • Use a gradient of increasing salt concentration for elution

  4. Size exclusion chromatography:

     • Final polishing step to obtain monodispersed protein

     • Also separates oligomeric forms if needed for specific applications

  5. Quality control:

     • SDS-PAGE to verify purity

     • Western blot with anti-MGM101 antibody to confirm identity

     • UV spectroscopy to assess DNA contamination (A260/A280 ratio)

     • Dynamic light scattering to verify homogeneity

  This protocol typically yields approximately 0.87mg of purified protein per liter of bacterial culture with minimal DNA contamination .

• How can one assess the functional activity of purified MGM101?

  Several assays can be employed to assess the functional activity of purified MGM101:

  1. DNA binding assays:

     • Electrophoretic mobility shift assay (EMSA) with single-stranded DNA substrates

     • Fluorescence anisotropy to measure binding kinetics and affinity

     • Surface plasmon resonance for real-time binding analysis

  2. DNA annealing activity:

     • Monitor the annealing of complementary single-stranded DNA molecules

     • Use a fluorescence-based assay with labeled oligonucleotides

     • Measure annealing with and without the mitochondrial ssDNA-binding protein (e.g., Rim1)

  3. Oligomerization analysis:

     • Cross-linking experiments using DSP (dithiobis[succinimidylpropionate])

     • Size exclusion chromatography to analyze oligomeric state

     • Electron microscopy to visualize ring and filament structures

  4. Functional complementation:

     • Transform MGM101-deficient yeast strains with the purified protein

     • Assess rescue of mtDNA stability phenotypes

     • Measure mitochondrial function recovery

  5. ATP-independent recombinase activity:

     • Assay for promoting homologous pairing between ssDNA and dsDNA duplex

     • Compare with known recombinases like Mhr1

  These assays collectively provide a comprehensive assessment of MGM101's structural integrity and biochemical activities related to its role in mtDNA maintenance.

• What are the key differences between MGM101 and other mitochondrial genome maintenance proteins?

  MGM101 has several distinctive features compared to other mitochondrial genome maintenance proteins:

  1. Structural characteristics:

     • Forms large oligomeric rings with ~14-fold symmetry

     • Can assemble into highly compressed helical filaments

     • Belongs to the Rad52-type recombination protein family

  2. Functional distinctions:

     Protein	Primary Function	DNA Binding Preference	ATP Requirement	Oligomeric State
     MGM101	ssDNA annealing, recombinational repair	Single-stranded DNA	ATP-independent	Large rings (~14-mer)
     Mhr1	Homologous pairing between ssDNA and dsDNA	Both ssDNA and dsDNA	ATP-independent	Unknown
     Rim1	ssDNA binding/protection	Single-stranded DNA	-	Tetramer
     Abf2p	mtDNA packaging	Double-stranded DNA	-	Monomer

  3. Evolutionary origin:

     • MGM101 appears to be of bacteriophage origin

     • Other maintenance proteins like Abf2p are related to nuclear HMG-box proteins

  4. Essentiality variation:

     • In S. cerevisiae, MGM101 is non-essential for cellular function aside from mitochondrial genome maintenance

     • In K. lactis, MGM101 is essential for viability of wild-type cells

     • Essentiality in D. hansenii is still being investigated

  5. Localization patterns:

     • MGM101 shows subnucleoid localization, present only in a subpopulation of nucleoids

     • Associates with the mitochondrial replisome spanning both mitochondrial membranes

     • Other proteins like Abf2p are more uniformly distributed across nucleoids

  Understanding these differences is crucial for elucidating the specific role of MGM101 within the complex machinery of mitochondrial genome maintenance.