Recombinant Mannheimia succiniciproducens Trigger factor (tig)

What is the role of Trigger Factor in M. succiniciproducens protein folding?

Trigger Factor (TF) in M. succiniciproducens functions as a ribosome-associated molecular chaperone with peptidyl-prolyl cis-trans isomerase (PPIase) activity. It assists in the proper folding of nascent polypeptides as they emerge from the ribosome, preventing premature aggregation or misfolding. Unlike other chaperones that require ATP for their function, TF operates in an ATP-independent manner. The protein binds directly to the 50S ribosomal subunit near the polypeptide exit tunnel, creating a protected environment for the emerging polypeptide chain. In M. succiniciproducens, TF likely plays a crucial role in maintaining proteostasis during growth and succinic acid production conditions, particularly helping fold metabolic enzymes involved in central carbon metabolism .

How is the tig gene structured and what are its key domains?

The tig gene in M. succiniciproducens encodes Trigger Factor, which contains three distinct functional domains:

• N-terminal domain (NTD): Responsible for ribosome binding through a conserved motif (often FRK) that interacts with the 50S ribosomal subunit

• Middle domain: Contains the PPIase activity and belongs to the FK506-binding protein family

• C-terminal domain: Forms an arm-like structure that creates a hydrophobic cavity for substrate binding

What expression systems are optimal for recombinant M. succiniciproducens Trigger Factor?

For efficient expression of recombinant M. succiniciproducens Trigger Factor, the following expression systems have proven effective:

• Vector systems:

  • pET vectors with T7 promoter for high-level expression

  • pAED4 vector system for controlled expression with IPTG induction

  • pACYC-based vectors with chloramphenicol resistance for moderate expression

• Expression conditions:

  • Host: E. coli BL21(DE3) or derivatives lacking proteases

  • Temperature: 20-25°C (lower temperatures improve soluble yield)

  • Induction: Mid-log phase (OD600 0.6-0.8) with 0.1-1.0 mM IPTG

  • Duration: 12-16 hours at reduced temperature

• Design considerations:

  • C-terminal His-tag preferable for purification

  • Codon optimization may improve expression

  • Consider co-expression with GroEL-GroES for enhanced solubility

The approach of using compatible plasmids that allow controlled expression of TF either alone or together with the GroEL-GroES chaperones has proven particularly effective for producing soluble, functional TF protein .

What is the recommended purification protocol for M. succiniciproducens Trigger Factor?

A systematic purification protocol for M. succiniciproducens Trigger Factor should include:

Step 1: Cell lysis

• Buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, protease inhibitors

• Method: Sonication or high-pressure homogenization

• Addition of DNase I (5 μg/ml) and MgCl₂ (5 mM) to reduce viscosity

Step 2: Initial purification

• Ni-NTA affinity chromatography for His-tagged TF

• Progressive washing with 20-40 mM imidazole

• Elution with 250-300 mM imidazole

Step 3: Secondary purification

• Size exclusion chromatography (Superdex 200)

• Alternative: Ion exchange chromatography (Q-Sepharose)

Step 4: Buffer exchange and storage

• Final buffer: 20 mM HEPES (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 5% glycerol

• Storage at -80°C in small aliquots

This protocol typically yields 20-30 mg of purified TF per liter of bacterial culture with >95% purity as assessed by SDS-PAGE. Functionality should be verified through PPIase activity assays using substrates like N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide .

How should researchers assess the quality and activity of purified TF?

A comprehensive quality assessment of purified M. succiniciproducens Trigger Factor should include:

Purity analysis:

• SDS-PAGE (target: >95% purity)

• Mass spectrometry to confirm molecular weight and detect potential modifications

Structural integrity:

• Circular dichroism spectroscopy to analyze secondary structure

• Thermal shift assays to determine protein stability

• Dynamic light scattering to assess aggregation state

Functional assays:

• PPIase activity using chromogenic substrates

• Ribosome binding assays using purified ribosomes

• Chaperone activity through prevention of model substrate aggregation

Activity metrics:

• Specific PPIase activity (μmol substrate converted/min/mg protein)

• Concentration-dependent activity analysis

• Comparison with E. coli TF as reference standard

Typical PPIase activity values for properly folded TF range from 10-20 mU/mg protein . Studies with Psychromonas arctica TF demonstrated that the purified protein displays functional PPIase activity in a concentration-dependent manner, which can serve as a good benchmark for M. succiniciproducens TF activity assessment .

How does M. succiniciproducens Trigger Factor recognize and bind substrate proteins?

M. succiniciproducens Trigger Factor recognizes and binds substrate proteins through a promiscuous mechanism that involves several key features:

• Substrate recognition patterns:

  • Preferential binding to hydrophobic amino acid stretches

  • Accommodation of diverse protein substrates with different structural properties

  • Formation of a cradle-like structure that can accommodate unfolded or partially folded proteins

• Binding interface:

  • Main binding site formed by the N-terminal and C-terminal domains creating a hydrophobic cavity

  • Multiple contact points allowing for accommodation of various substrate sizes

  • Dynamic binding enabling progressive folding of substrates

• Substrate diversity:

  • Proteomic studies with E. coli TF identified over 170 cytosolic proteins as potential substrates

  • Ribosomal proteins (like S7) are well-characterized TF substrates

  • Both co-translational and post-translational binding capabilities

The crystal structure of TF in complex with ribosomal protein S7 from Thermotoga maritima has provided valuable insights into substrate recognition, revealing the molecular basis for TF's ability to accommodate diverse protein substrates . Similar mechanisms likely apply to M. succiniciproducens TF based on structural conservation of this chaperone across bacterial species.

How does Trigger Factor cooperate with other chaperone systems?

Trigger Factor in M. succiniciproducens likely operates within a complex chaperone network similar to that observed in other bacteria:

• Sequential chaperone pathway:

  • TF acts as first-line chaperone for nascent chains emerging from ribosomes

  • Proteins requiring additional assistance are handed over to downstream chaperones (GroEL-GroES, DnaK-DnaJ-GrpE)

  • Some proteins require only TF while others need multiple chaperones

• Functional overlap with DnaK system:

  • TF and DnaK show partially overlapping substrate specificity

  • Deletion of both tig and dnaK genes can create synthetic lethality under standard conditions

  • Survival is possible under specific growth conditions (e.g., lower temperatures)

• Synergy with GroEL-GroES:

  • TF can work synergistically with GroEL-GroES for certain substrates

  • For some aggregation-prone proteins, co-expression of TF with GroEL-GroES is more effective than either chaperone alone

  • TF may strengthen GroEL-substrate binding to facilitate proper folding

This cooperative network ensures robust protein folding under various conditions, with TF playing a central role in initial folding events. The table below summarizes experimental observations on chaperone cooperation effects:

Data derived from studies with E. coli TF co-expression systems

What role does Trigger Factor play in cold adaptation?

Trigger Factor plays a significant role in cold adaptation in bacteria, with potential implications for M. succiniciproducens:

• Enhanced importance at lower temperatures:

  • Protein folding is inherently slower and more challenging at reduced temperatures

  • TF activity becomes particularly critical under these conditions

  • Studies with psychrophilic bacteria (growing at 4-15°C) demonstrate that their TF proteins help cells survive cold environments

• Functional adaptations:

  • Cold-adapted TF variants may exhibit higher catalytic efficiency at lower temperatures

  • Research with Psychromonas arctica showed that expressed PaTF proteins helped cells survive against cold treatment at 4°C

  • The purified PaTF displayed functional PPIase activity in a concentration-dependent manner

• Experimental observations:

  • TF expression may be upregulated at lower temperatures

  • TF from psychrophilic bacteria demonstrates adaptive features compared to mesophilic homologs

  • These adaptations could inform engineering of M. succiniciproducens TF for applications requiring low-temperature activity

Understanding these cold-adaptive features could be particularly relevant for industrial applications of M. succiniciproducens involving reduced-temperature fermentation conditions to minimize by-product formation during succinic acid production.

How can M. succiniciproducens Trigger Factor enhance soluble protein production?

M. succiniciproducens Trigger Factor can significantly improve soluble protein production through several strategic approaches:

• Co-expression strategies:

  • Dual-plasmid systems with compatible origins of replication

  • Controlled expression systems for TF either alone or with GroEL-GroES

  • Sequential induction with TF expression preceding target protein induction

• Target protein considerations:

  • TF is especially effective for proteins with:

    • High proline content requiring isomerization

    • Hydrophobic regions prone to aggregation

    • Complex multi-domain structures

• Experimental evidence of efficacy:

  • Studies with E. coli TF demonstrated marked effects on preventing aggregation of proteins including:

    • Mouse endostatin (20 kDa)

    • Human oxygen-regulated protein ORP150 (150 kDa)

    • Human lysozyme (14 kDa)

• Synergistic approaches:

  • For complex target proteins, co-expression of TF with GroEL-GroES often produces superior results

  • This combination has demonstrated synergistic effects on protein folding for certain substrates

  • For some targets, TF alone is sufficient (e.g., endostatin), while others benefit from combined chaperone systems

The impact of TF on soluble fraction recovery can be substantial, as shown in published data where proteins primarily found in the insoluble fraction were recovered in the soluble fraction upon TF co-expression .

What experimental design is recommended for optimizing TF-assisted protein folding?

A systematic experimental design for optimizing Trigger Factor-assisted protein folding should include:

Phase 1: Baseline establishment

• Express target protein alone under standard conditions

• Analyze soluble vs. insoluble fractions quantitatively

• Document aggregation behavior and yields

Phase 2: TF co-expression optimization

• Test multiple TF expression vectors with varying promoter strengths

• Evaluate timing strategies:

  • Pre-express TF before target induction

  • Simultaneous induction

  • Gradual induction using titratable systems

• Compare TF alone vs. TF with other chaperones (GroEL-GroES, DnaK-DnaJ-GrpE)

Phase 3: Condition optimization matrix

• Temperature (15°C, 20°C, 25°C, 30°C, 37°C)

• Inducer concentrations (0.01 mM to 1.0 mM IPTG)

• Media formulations (standard LB, enriched media, minimal media)

• Cell density at induction (early/mid/late log phase)

Phase 4: Analysis

• Quantitative solubility analysis (densitometry of SDS-PAGE gels)

• Specific activity measurements of target protein

• Structural characterization (CD spectroscopy, limited proteolysis)

• Aggregation monitoring (light scattering, filter retention)

This tiered approach allows systematic identification of optimal conditions for TF-assisted folding of specific target proteins. Real-time monitoring of protein folding using fluorescent protein fusions can provide additional insights into the kinetics of folding enhancement.

How does M. succiniciproducens Trigger Factor compare with TF from other species?

Comparative analysis of M. succiniciproducens Trigger Factor with TF from other bacterial species reveals important considerations for recombinant protein production:

• Functional comparison across species:

  • E. coli TF: Most extensively studied; serves as the reference standard

  • Psychrophilic bacteria TF (e.g., Psychromonas arctica): Enhanced activity at lower temperatures

  • Thermophilic bacteria TF (e.g., Thermotoga maritima): Higher stability during purification

  • M. succiniciproducens TF: Potentially specialized for optimal folding in this organism's metabolic context

• Structural determinants of function:

  • Sequence alignments reveal conserved functional domains but species-specific adaptations

  • Ribosome binding affinity may differ between species

  • Substrate specificity profiles vary based on evolutionary adaptations

  • PPIase activity levels and temperature dependence differ among species

• Selection criteria for applications:

  • Temperature requirements for target protein expression

  • Complexity of target protein folding pathway

  • Source organism of target protein (matching TF to source may improve compatibility)

  • Scale and purpose of recombinant protein production

For applications involving moderate temperature expression with minimal by-product formation, M. succiniciproducens TF may offer advantages due to the organism's natural metabolic efficiency in producing succinic acid with minimal by-products .

What challenges exist in expressing and purifying functionally active TF?

Researchers face several key challenges when expressing and purifying functionally active M. succiniciproducens Trigger Factor:

• Expression challenges:

  • Potential toxicity at high expression levels (as observed with E. coli TF)

  • Proper folding of TF itself may require chaperone assistance

  • Balancing yield and proper folding at different temperatures

  • Codon usage optimization for heterologous expression

• Purification hurdles:

  • Preventing aggregation during cell lysis and purification

  • Separating TF from bound substrate proteins

  • Maintaining native conformation throughout purification

  • Preserving ribosome-binding capability

• Activity preservation:

  • Ensuring integrity of all three functional domains

  • Maintaining PPIase activity, which can be sensitive to buffer conditions

  • Preserving proper oligomeric state (TF can form dimers off the ribosome)

  • Protecting against oxidation of critical residues

• Quality control:

  • Distinguishing between properly folded and misfolded TF

  • Establishing reliable activity assays specific for M. succiniciproducens TF

  • Determining appropriate storage conditions

Studies with E. coli TF have shown that even modest overexpression (approximately fourfold) can be toxic to cells, suggesting careful titration of expression levels is crucial . Additionally, high levels of TF can lead to specific protein aggregation, as demonstrated by the cytosolic accumulation of pre-OmpF when TF was overexpressed in E. coli .

How can researchers investigate TF substrate specificity?

Investigating the substrate specificity of M. succiniciproducens Trigger Factor requires a multi-faceted approach:

• Proteome-wide substrate identification:

  • Pull-down experiments using tagged TF followed by mass spectrometry

  • Comparative analysis of protein aggregation in TF-depleted vs. TF-overexpressing strains

  • Ribosome-nascent chain complex isolation to identify co-translational substrates

• Biochemical characterization:

  • Surface plasmon resonance to measure binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Structural analysis approaches:

  • X-ray crystallography of TF with model peptides

  • Cryo-EM to visualize TF interaction with larger substrates

  • NMR spectroscopy to identify dynamic binding regions

• Comparative profiling:

  • Analysis of binding to proteins from various structural classes

  • Comparison with substrate preferences of other bacterial TFs

  • Investigation of organism-specific substrates reflecting M. succiniciproducens metabolism

Research with E. coli TF identified over 170 cytosolic proteins as TF substrates, including ribosomal protein S7, which has been characterized in detail with TF from Thermotoga maritima . Similar approaches could reveal the M. succiniciproducens TF substrate landscape.

What are the implications of TF research for M. succiniciproducens metabolic engineering?

Research on Trigger Factor in M. succiniciproducens has significant implications for metabolic engineering efforts:

• Support for metabolic enzymes:

  • TF ensures proper folding of key enzymes in the succinate production pathway

  • Critical enzymes potentially requiring TF assistance include:

    • Phosphoenolpyruvate carboxykinase (PckA)

    • Fumarase (FumC)

    • Malate dehydrogenase

  • Proper folding directly impacts carbon flux toward succinate

• Sugar transport system folding:

  • M. succiniciproducens utilizes phosphotransferase systems (PTS) for sugar uptake

  • TF may assist in folding PTS components including phosphocarrier protein HPr and sugar-specific IIA components

  • Efficient carbon source uptake is prerequisite for high succinate yields

• Stress adaptation during fermentation:

  • Industrial fermentation imposes various stresses (pH shifts, metabolite accumulation)

  • TF contributes to maintaining proteome integrity under stress conditions

  • Enhanced stress resistance could improve succinate production robustness

• Engineering approaches:

  • TF co-expression could stabilize engineered metabolic pathways

  • Modulating TF levels might optimize enzyme expression for key pathway steps

  • TF variants could be developed for improved folding of rate-limiting enzymes

Proteomic studies have shown that when Mg(OH)₂ was employed as a neutralizing agent during M. succiniciproducens fermentation, enzymes involved in succinate production showed significantly increased levels. Specifically, PckA exhibited fold changes of up to 7.11 during high growth rate and 4.38 during zero growth rate conditions, while FumC showed increases of 10.82 and 1.64 fold, respectively . TF likely plays a role in ensuring the proper folding of these critical enzymes.

What analytical methods are recommended for characterizing PPIase activity?

Characterizing the peptidyl-prolyl isomerase (PPIase) activity of M. succiniciproducens Trigger Factor requires specialized analytical methods:

• Standard chromogenic assays:

  • Coupled chymotrypsin assay using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide:

    • Protocol: PPIase activity converts substrate from cis to trans configuration

    • Only the trans form is cleaved by chymotrypsin, releasing p-nitroaniline

    • Activity measured as absorbance increase at 390-400 nm

  • Typical conditions:

    • Buffer: 35 mM HEPES, pH 7.8

    • Temperature: 10-15°C

    • Substrate concentration: 60-100 μM

    • Chymotrypsin: 50-100 μg/ml

• Advanced spectroscopic methods:

  • NMR spectroscopy for real-time monitoring of cis/trans isomerization

  • Fluorescence-based assays with specially designed peptide substrates

  • Circular dichroism to monitor secondary structure changes during isomerization

• Enzyme kinetic parameters:

  • Determination of Km and Vmax across different temperatures

  • Calculation of catalytic efficiency (kcat/Km)

  • Testing with physiologically relevant substrates derived from M. succiniciproducens proteins

• Controls and validation:

  • Inhibitor studies using FK506 or rapamycin

  • Comparison with PPIase-deficient TF mutants

  • Benchmark against E. coli TF as reference standard

Studies with purified Psychromonas arctica TF demonstrated functional PPIase activity that increased in a concentration-dependent manner . Similar concentration-dependent assays should be conducted with M. succiniciproducens TF to establish its specific activity profile.

How does Trigger Factor contribute to M. succiniciproducens succinic acid production?

Trigger Factor contributes to M. succiniciproducens succinic acid production through several critical mechanisms:

• Support for central metabolic pathways:

  • Proper folding of key enzymes in the succinic acid production pathway

  • Maintenance of enzyme stability during fermentation

  • Potentially enhanced activity of critical enzymes through co-translational folding assistance

• Role in carbon source utilization:

  • M. succiniciproducens utilizes various carbon sources including sucrose via a phosphotransferase system (PTS)

  • TF may assist in folding PTS components, enhancing carbon uptake efficiency

  • Improved carbon source utilization directly impacts succinic acid yields

• Stress resistance during production:

  • High-titer succinic acid production creates stressful conditions

  • TF contributes to maintaining protein homeostasis during pH fluctuations and metabolite accumulation

  • Improved stress tolerance translates to more robust production capabilities

The engineering of M. succiniciproducens for succinic acid production has achieved remarkable results, with recent advances reporting production of 152.23 ± 0.99 g/L of succinic acid with a yield of 1.30 ± 0.01 mol/mol glucose equivalent . The robustness of the protein folding machinery, including TF, likely contributes significantly to achieving such high production levels.