Recombinant Mannheimia succiniciproducens Magnesium transport protein CorA (corA)

What is Mannheimia succiniciproducens and why is it significant for research?

Mannheimia succiniciproducens is a capnophilic succinic acid-producing bacterium originally isolated from the rumens of Korean cows. Its significance stems from its remarkable ability to produce large amounts of succinic acid under anaerobic conditions in the presence of CO₂. The complete genome sequence of M. succiniciproducens has been determined, enabling in-silico genome-scale metabolic analysis and targeted metabolic engineering approaches . This bacterium has become an important model organism for studying fermentative metabolism and for developing metabolically engineered strains capable of producing succinic acid without by-product formation. Through genome-based metabolic engineering, researchers have successfully eliminated major pathways that lead to by-product formation by disrupting genes such as ldhA, pflB, pta, and ackA .

How is recombinant M. succiniciproducens CorA protein typically expressed?

Recombinant M. succiniciproducens CorA protein is typically expressed in Escherichia coli expression systems. According to commercial product information, the full-length protein (amino acids 1-316) is expressed with an N-terminal His-tag to facilitate purification . The expression process involves:

• Cloning the corA gene into a suitable expression vector

• Transforming the recombinant vector into an E. coli expression strain

• Inducing protein expression under optimized conditions

• Lysing the cells and purifying the protein using affinity chromatography

• Processing the purified protein into a lyophilized powder for storage and distribution

This approach leverages E. coli's advantages as an expression host, including its rapid growth, ease of genetic manipulation, and cost-effectiveness . The final recombinant protein product is typically stored in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 to maintain stability .

What are the recommended storage conditions for recombinant CorA protein?

Based on commercial product specifications, the following storage conditions are recommended for maintaining the stability and functionality of recombinant M. succiniciproducens CorA protein:

Proper aliquoting upon receipt is essential to prevent protein degradation from repeated freeze-thaw cycles . For applications requiring long-term storage, adding glycerol to a final concentration of 50% after reconstitution is recommended to maintain protein stability .

What cloning strategies are most effective for generating recombinant M. succiniciproducens proteins?

Several cloning strategies have proven effective for generating recombinant bacterial proteins, including those from M. succiniciproducens. Each approach offers distinct advantages depending on research requirements:

• Recombination-based cloning: Systems like Gateway (Thermo Fisher Scientific) utilize site-specific recombination to construct vectors without restriction enzymes and ligases. The Gateway system exploits bacteriophage λ's site-specific recombination system to shuttle sequences between plasmids with compatible recombination attachment (att) sites . Once an entry clone is created, the gene can be easily subcloned into various destination vectors using the LR reaction, making it ideal for high-throughput approaches .

• Seamless Ligation Cloning Extract (SLiCE) method: This cost-effective approach uses cell extracts from modified E. coli strains expressing an optimized λ prophage Red recombination system. Homemade SLiCE can be prepared from common RecA- E. coli laboratory strains like DH5α or JM109 at a cost of approximately $0.003 per reaction, making it suitable for high-throughput cloning applications .

• Cold Fusion and CloneEZ systems: These commercial kits offer alternatives for assembly of DNA fragments into vectors in single reactions .

For membrane proteins like CorA, vectors containing inducible promoters (such as T7) and fusion tags that enhance solubility are particularly beneficial. The choice of cloning strategy should consider factors including cost, efficiency, sequence flexibility, and downstream expression requirements.

What expression systems optimize yields of functional recombinant CorA protein?

Optimizing the expression of functional recombinant M. succiniciproducens CorA protein requires careful consideration of several factors:

Expression Host Selection:

• E. coli strains: BL21(DE3) derivatives are commonly used for membrane protein expression due to their reduced protease activity

• Specialized strains: C41(DE3) or C43(DE3) were developed specifically for expressing toxic or membrane proteins

• Codon-optimized strains: Rosetta or CodonPlus strains can supply rare tRNAs if codon bias is a concern

Expression Vector Considerations:

• Promoter strength: T7 or tac promoters offer strong, inducible expression

• Fusion partners: His-tags facilitate purification while larger tags like MBP or SUMO can enhance solubility

• Secretion signals: For potential periplasmic or extracellular expression if cytoplasmic expression forms inclusion bodies

Optimization Parameters:

• Induction temperature: Lower temperatures (15-25°C) often improve membrane protein folding

• Inducer concentration: Titrating IPTG or other inducers to find optimal levels

• Expression time: Extended expression at lower temperatures may yield more functional protein

• Media composition: Enhanced media formulations or supplementation with specific ions like Mg²⁺

For membrane proteins like CorA, optimizing extraction and purification conditions using appropriate detergents is equally critical to maintain the protein's native conformation and functionality after expression.

What purification strategies are most effective for His-tagged M. succiniciproducens CorA?

Purifying His-tagged M. succiniciproducens CorA requires specialized strategies that account for its membrane protein nature. The following purification workflow has proven effective:

Primary Purification:

• Cell lysis optimization: Gentle lysis methods using specialized buffers containing membrane-protein-compatible detergents like n-dodecyl-β-D-maltoside (DDM) or CHAPS

• Immobilized Metal Affinity Chromatography (IMAC): Using Ni-NTA or Co-NTA resins with optimized binding, washing, and elution conditions

• Buffer composition: Including stabilizing agents such as glycerol (5-10%) and appropriate salt concentrations (typically 100-300 mM NaCl)

Secondary Purification:

• Size Exclusion Chromatography (SEC): To remove aggregates and separate oligomeric states

• Ion Exchange Chromatography: As a polishing step if higher purity is required

Critical Considerations:

• Maintaining detergent concentration above critical micelle concentration throughout purification

• Including magnesium (typically 1-5 mM MgCl₂) in buffers to stabilize the protein

• Avoiding harsh elution conditions that might denature the protein

• Considering on-column refolding if inclusion bodies form

• Monitoring protein quality using techniques like dynamic light scattering or SEC-MALS

This strategic approach maximizes both yield and biological activity of the purified CorA protein, crucial for downstream functional and structural studies.

What are the most common challenges in expressing recombinant CorA and how can they be overcome?

Researchers frequently encounter several challenges when expressing recombinant M. succiniciproducens CorA protein. The following table outlines these challenges and provides tested solutions:

Understanding the membrane protein nature of CorA and applying these targeted solutions can significantly improve expression outcomes and yield functional protein for downstream applications.

What are the structural characteristics of M. succiniciproducens CorA and how do they relate to function?

The M. succiniciproducens CorA protein shares key structural features with other bacterial CorA transporters, though specific details may vary. Based on the amino acid sequence and comparison with homologous proteins, the following structural characteristics can be inferred:

Domain Organization:

• A large N-terminal cytoplasmic domain (approximately two-thirds of the protein)

• A transmembrane domain with a specific architecture likely consisting of two transmembrane helices near the C-terminus

• A signature GMN motif (visible in the sequence as "GMNFEFMPE") that is highly conserved across CorA proteins and critical for magnesium selectivity

Functional Elements:

• The cytoplasmic domain likely contains divalent cation-sensing regions that regulate transport activity

• The transmembrane domain forms the ion conduction pathway

• Specific residues in the pore region determine ion selectivity and transport kinetics

The protein likely functions as a homopentamer, forming a symmetrical channel through the membrane. The mechanism of transport involves conformational changes triggered by intracellular magnesium levels, where high concentrations cause the channel to close through allosteric regulation.

The sequence (MINAFALENARLTRLDEDNLSTLNKAIWIDLVEPTSEEREILQDGLEQSLASFLELE...) reveals charged and polar residues that likely contribute to ion coordination during transport, while the transmembrane regions contain more hydrophobic residues essential for membrane integration .

What functional assays are recommended to verify CorA magnesium transport activity?

To verify the functional activity of recombinant M. succiniciproducens CorA as a magnesium transporter, researchers should consider the following complementary assays:

In vitro Transport Assays:

• Liposome-based flux assays: Reconstituting purified CorA into liposomes and measuring Mg²⁺ uptake using:

  • Magnesium-sensitive fluorescent dyes (Mag-fura-2, Magnesium Green)

  • Isotope-labeled magnesium (²⁸Mg²⁺)

  • Atomic absorption spectroscopy to quantify transported magnesium

• Electrophysiological measurements:

  • Patch-clamp recordings of CorA reconstituted into giant unilamellar vesicles or planar lipid bilayers

  • Solid-supported membrane electrophysiology for measuring charge movement during transport

Binding and Conformational Change Assays:

• Isothermal Titration Calorimetry (ITC): To determine the binding affinity and thermodynamics of Mg²⁺ interactions with CorA

• Thermal shift assays: To detect stabilization of the protein structure upon Mg²⁺ binding

• Circular Dichroism spectroscopy: To monitor structural changes upon substrate binding

Cellular Complementation Assays:

• Functional complementation: Testing if M. succiniciproducens CorA can restore growth of CorA-deficient bacterial strains under magnesium-limited conditions

• Growth inhibition assays: Using cobalt sensitivity as a proxy for CorA activity (since Co²⁺ is toxic and can be transported by CorA)

A comprehensive assessment should combine multiple approaches to confirm both the magnesium binding capacity and transport functionality of the recombinant protein.

How can researchers determine the kinetic parameters of CorA-mediated magnesium transport?

Determining the kinetic parameters of CorA-mediated magnesium transport requires rigorous experimental design and careful data analysis. The following methodology will yield reliable kinetic measurements:

Experimental Design:

• Reconstitution system optimization:

  • Establish a consistent proteoliposome preparation with verified CorA orientation

  • Control lipid composition to mimic bacterial membrane environments

  • Determine protein-to-lipid ratios that provide measurable transport without aggregation

• Transport measurement setup:

  • Use rapid kinetic techniques (stopped-flow fluorescence, quenched-flow)

  • Establish linear initial rate conditions at various substrate concentrations

  • Include appropriate controls (protein-free liposomes, heat-inactivated protein)

Kinetic Analysis:

• Michaelis-Menten kinetics determination:

  • Measure initial transport rates at multiple Mg²⁺ concentrations (typically 0.01-10 mM)

  • Plot transport rate versus substrate concentration

  • Fit data to the Michaelis-Menten equation: v = (Vmax × [S])/(Km + [S])

  • Extract Km (apparent affinity for Mg²⁺) and Vmax (maximum transport rate)

• Advanced kinetic analysis:

  • Analyze potential cooperativity using Hill plots

  • Examine substrate inhibition effects at high Mg²⁺ concentrations

  • Determine the effects of membrane potential on transport kinetics

• Inhibition studies:

  • Test competitive inhibition by other divalent cations (Ca²⁺, Co²⁺, Ni²⁺)

  • Calculate inhibition constants (Ki) for various inhibitors

This systematic approach provides a comprehensive kinetic profile of the recombinant CorA protein, enabling comparison with homologous transporters and correlation with structural features.

Potential Species-Specific Adaptations:

• M. succiniciproducens is a capnophilic organism optimized for succinic acid production, suggesting possible adaptations in its ion transport systems to support this specialized metabolism

• The organism's adaptation to the rumen environment may have shaped the kinetic properties of its CorA protein, potentially optimizing transport rates for this ecological niche

• Sequence variations in key regions may affect:

  • Transport kinetics and Mg²⁺ affinity

  • Sensitivity to inhibition by other divalent cations

  • Regulatory responses to environmental conditions

Comparative functional studies between M. succiniciproducens CorA and homologs from model organisms like E. coli or well-characterized extremophiles like T. maritima would provide valuable insights into how this essential transporter has evolved to support the specialized metabolism of this industrially important bacterium.

How can recombinant M. succiniciproducens CorA be used for structural biology studies?

Recombinant M. succiniciproducens CorA protein provides an excellent subject for structural biology investigations, offering insights into both magnesium transport mechanisms and membrane protein structure-function relationships. The following approaches represent state-of-the-art methodologies for structural characterization:

X-ray Crystallography:

• Optimize detergent screening for crystal formation (using techniques like vapor diffusion or lipidic cubic phase)

• Consider fusion partners that enhance crystallizability (T4 lysozyme, BRIL)

• Utilize heavy atom derivatives or selenomethionine incorporation for phase determination

• Explore crystallization in the presence and absence of magnesium to capture different conformational states

Cryo-Electron Microscopy:

• Prepare homogeneous samples in detergent micelles, nanodiscs, or amphipols

• Apply single-particle analysis for high-resolution structure determination

• Use time-resolved cryo-EM to potentially capture transport intermediates

• Leverage direct electron detectors and advanced image processing for high-resolution details

Complementary Structural Techniques:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamics and ligand-binding regions

• Small-angle X-ray scattering (SAXS) for solution-state structural information

• Solid-state NMR to investigate specific residues involved in magnesium coordination

• Site-directed spin labeling with EPR spectroscopy to measure distances between specific residues

These structural studies would provide critical insights into the magnesium transport mechanism, potentially revealing conformational changes that occur during transport and the molecular basis for ion selectivity.

How might recombinant M. succiniciproducens CorA contribute to understanding magnesium homeostasis in industrial fermentation?

Recombinant M. succiniciproducens CorA protein offers a valuable tool for investigating magnesium homeostasis in industrial fermentation processes, particularly for succinic acid production. This research direction has several significant implications:

Metabolic Engineering Applications:

• Magnesium ions serve as essential cofactors for many enzymes in the succinic acid production pathway, including phosphoenolpyruvate (PEP) carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase

• Understanding CorA's role in magnesium uptake could inform strategies to optimize intracellular magnesium levels for maximum enzyme activity

• Engineering CorA expression levels might enhance succinic acid production by ensuring optimal magnesium availability

Process Optimization Insights:

• Characterizing how CorA activity responds to fermentation conditions (pH, temperature, ionic strength) could guide bioprocess optimization

• Identifying the optimal extracellular magnesium concentration for maximum transport efficiency

• Understanding how transport kinetics change during different growth phases could inform feeding strategies

Integration with Metabolic Models:

This research direction directly connects fundamental studies of a membrane transporter to applied bioprocessing, potentially leading to improvements in the industrial production of succinic acid, which has been reported to reach yields of 134.25 g/L with a productivity of 21.3 g/L/h in optimized strains .

What emerging technologies are advancing the study of membrane transporters like M. succiniciproducens CorA?

The field of membrane transporter research is rapidly evolving, with several cutting-edge technologies offering new opportunities for studying proteins like M. succiniciproducens CorA:

Advanced Structural Methods:

• Cryo-electron tomography: Enables visualization of membrane proteins in their native cellular context, potentially revealing how CorA organizes within the bacterial membrane

• Micro-electron diffraction (MicroED): Allows structure determination from microcrystals too small for traditional X-ray crystallography

• Serial femtosecond crystallography: Uses X-ray free-electron lasers to obtain structures from microcrystals without radiation damage

Single-Molecule Techniques:

• Single-molecule FRET (smFRET): Monitors real-time conformational changes in individual CorA molecules during transport cycles

• Nanopore recording: Measures ion flow through individual transporter molecules incorporated into artificial membranes

• High-speed atomic force microscopy (HS-AFM): Visualizes structural dynamics of membrane proteins at the nanoscale with temporal resolution

Innovative Expression and Reconstitution Systems:

• Cell-free protein synthesis: Enables direct integration of synthesized CorA into nanodiscs or liposomes

• Synthetic cell systems: Reconstitutes transport function in minimal cell models with defined composition

• DNA origami scaffolds: Creates precisely defined membrane environments for transporter studies

Computational Approaches:

• Machine learning for structure prediction: Tools like AlphaFold2 can predict structures of transporters with increasing accuracy

• Enhanced sampling molecular dynamics: Simulates rare transport events that occur on timescales inaccessible to conventional simulations

• Graph neural networks: Predicts functional effects of mutations based on structural and evolutionary data

These technologies collectively promise to provide unprecedented insights into the structure, dynamics, and function of membrane transporters like CorA, potentially revealing mechanistic details that have remained elusive with traditional approaches.

How can researchers troubleshoot expression issues with recombinant M. succiniciproducens CorA?

When encountering expression challenges with recombinant M. succiniciproducens CorA, researchers should implement a systematic troubleshooting approach. The following decision tree addresses common issues and provides evidence-based solutions:

Low or No Expression:

• Verify construct integrity:

  • Confirm sequence correctness through DNA sequencing

  • Check reading frame and promoter regions

  • Ensure plasmid stability in expression host

• Optimize transcription:

  • Test alternative promoters with different strengths or induction mechanisms

  • Verify mRNA production levels through RT-PCR

  • Examine for potential toxic effects on host cells

• Enhance translation:

  • Optimize codon usage for E. coli

  • Improve ribosome binding site efficiency

  • Test different N-terminal fusion tags known to enhance expression

Protein Solubility Issues:

• Expression condition optimization:

  • Reduce induction temperature (15-20°C)

  • Lower inducer concentration

  • Test slower induction methods (auto-induction media)

• Solubility enhancement strategies:

  • Screen multiple solubility-enhancing fusion tags (MBP, SUMO, TrxA)

  • Co-express with molecular chaperones

  • Test specialized E. coli strains designed for membrane proteins (C41/C43)

Extraction and Purification Problems:

• Membrane protein-specific approaches:

  • Screen multiple detergents for extraction efficiency

  • Optimize detergent:protein ratios

  • Consider alternative solubilization methods (SMALPs, nanodiscs)

• Purification optimization:

  • Adjust imidazole concentrations in binding/washing/elution buffers

  • Include stabilizing additives (glycerol, specific salts, magnesium)

  • Test mild reducing agents if disulfide scrambling is suspected

This systematic approach addresses the most common issues encountered during recombinant expression of membrane proteins like CorA, providing specific, actionable solutions at each step of the troubleshooting process.

What strategies help optimize the solubility and stability of purified recombinant CorA protein?

Optimizing the solubility and stability of purified recombinant M. succiniciproducens CorA protein requires careful consideration of buffer components and storage conditions. The following evidence-based strategies have proven effective for membrane proteins like CorA:

Buffer Optimization:

• Detergent selection and concentration:

  • Screen detergents systematically (maltosides, glucosides, neopentyl glycols)

  • Maintain detergent concentration slightly above CMC

  • Consider detergent mixtures for improved stability

• Buffer composition:

  • Test pH range (typically 7.0-8.0 for optimal stability)

  • Optimize ionic strength (typically 100-300 mM NaCl)

  • Include stabilizing additives:

    • Glycerol (5-20%)

    • Trehalose (as used in commercial preparations at 6%)

    • Specific lipids that may interact with the protein

• Critical ion supplementation:

  • Add magnesium (1-5 mM) as the transported substrate

  • Test effects of other divalent cations at low concentrations

Storage and Handling:

• Physical stability enhancement:

  • Aliquot to prevent freeze-thaw cycles

  • Flash-freeze in liquid nitrogen

  • Store at -80°C for long-term or -20°C for medium-term storage

• Alternative stabilization approaches:

  • Transfer to more stable membrane mimetics (nanodiscs, amphipols)

  • Explore protein engineering of stabilizing mutations

  • Consider chemical crosslinking for structural studies

• Quality assessment methods:

  • Monitor stability over time using size-exclusion chromatography

  • Assess function periodically with transport assays

  • Use thermal shift assays to identify stabilizing conditions

The commercial preparation of recombinant M. succiniciproducens CorA uses a Tris/PBS-based buffer with 6% Trehalose at pH 8.0, suggesting these conditions provide good baseline stability . For reconstitution, addition of glycerol to 5-50% is recommended for long-term storage, with 50% being the standard concentration used commercially .