Recombinant Escherichia coli Protein trbH (trbH)

What is the optimal expression system for recombinant trbH protein production?

The optimal expression system for recombinant trbH protein involves using E. coli as the host organism with an N-terminal His-tag fusion. This approach facilitates efficient purification while maintaining protein functionality. The full-length trbH protein (239 amino acids) can be successfully expressed using standard E. coli expression vectors with appropriate promoters. For optimal results, use a Tris/PBS-based buffer system with 6% trehalose at pH 8.0, which enhances protein stability during storage and reconstitution .

When selecting expression systems, consider implementing a dual-promoter strategy. Studies have shown that gene expression can be significantly increased under the control of tandem promoters compared to single promoters in bacterial systems. This approach may elevate productivity by 11-12 fold compared to single promoter systems, potentially enhancing trbH yields .

What are the recommended storage conditions for recombinant trbH protein?

For optimal stability of recombinant trbH protein, store the lyophilized powder at -20°C to -80°C upon receipt. After reconstitution, the protein should be stored in working aliquots at 4°C for up to one week to avoid degradation. For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being optimal) before aliquoting and storing at -20°C or -80°C .

Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of activity. When reconstituting the protein, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. Prior to opening the vial, brief centrifugation is recommended to bring contents to the bottom .

How can I optimize the purification protocol for recombinant His-tagged trbH protein?

The purification of His-tagged trbH protein can be optimized through a multi-step approach:

• Immobilized Metal Affinity Chromatography (IMAC): Utilize Ni-NTA or Co-NTA resins with a carefully optimized imidazole gradient (20-250 mM) for elution. This initial step captures His-tagged trbH with approximately 85-90% purity.

• Buffer Optimization: Use buffers containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 5-10% glycerol to maintain protein stability throughout purification.

• Secondary Purification: Follow IMAC with size exclusion chromatography using a Superdex 75 or 200 column to achieve >95% purity. This step removes aggregates and improves homogeneity.

• Protein Verification: Confirm protein identity and purity using SDS-PAGE, which should show a predominant band at approximately 27-28 kDa (accounting for the His-tag) .

• Activity Assessment: Develop function-specific assays to verify that the purified protein maintains its biological activity after the purification process.

This systematic approach typically yields high-purity trbH protein suitable for various research applications, including structural studies and functional assays.

How can I enhance the production yield of recombinant trbH protein in E. coli?

Enhancing production yield of recombinant trbH protein can be achieved through multiple strategies:

• Co-expression of ftsA and ftsZ genes: Research has demonstrated that co-expressing these key cell division proteins can significantly improve both cell growth and recombinant protein production. This approach suppresses cell filamentation that commonly occurs during overproduction of recombinant proteins, allowing cultures to reach higher cell densities and increase volumetric productivity. Studies with other recombinant proteins showed up to 2-fold increase in volumetric productivity using this method .

• Implementing double promoter systems: The use of tandem promoters has been shown to significantly enhance recombinant protein expression. By constructing expression vectors with double promoters, protein yields can be increased compared to single promoter systems. This strategy has been successfully applied to various recombinant proteins in bacterial expression systems .

• Optimization of fed-batch cultivation: pH-stat fed-batch cultures have demonstrated superior results for recombinant protein production. In the case of other recombinant proteins expressed in E. coli TG1, cultures co-expressing ftsA and ftsZ genes achieved cell concentrations of 27.5 g DCW/liter compared to 17.5 g DCW/liter without co-expression, with specific growth rates of 0.13 h⁻¹ versus 0.10 h⁻¹ .

• Temperature modulation: Lowering cultivation temperature to 25-30°C after induction can reduce inclusion body formation and improve the yield of soluble protein, which may be beneficial for trbH expression.

What are the challenges in maintaining structural integrity of trbH during expression and purification?

Maintaining structural integrity of trbH during expression and purification presents several challenges:

• Inclusion body formation: trbH may form inclusion bodies during overexpression, particularly at high induction levels. This can be addressed by:

  • Lowering induction temperature to 16-25°C

  • Reducing inducer concentration

  • Co-expressing molecular chaperones such as GroEL/GroES or DnaK/DnaJ/GrpE systems

  • Using weaker promoters or lower copy number plasmids

• Protein aggregation during purification: The hydrophobic nature of certain regions in trbH may lead to aggregation during concentration steps. This can be mitigated by:

  • Including 5-10% glycerol in all purification buffers

  • Adding low concentrations (0.05-0.1%) of non-ionic detergents like Triton X-100

  • Maintaining protein solutions at concentrations below 1 mg/mL until final concentration steps

• Oxidation sensitivity: If trbH contains cysteine residues, oxidation can affect structural integrity. Prevention strategies include:

  • Adding reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to all buffers

  • Working under nitrogen atmosphere during critical purification steps

  • Including antioxidants like 1 mM EDTA in storage buffers

The amino acid sequence of trbH (MNRSTPVFNSQAAHTFKFPGVISHNNQSPTAGMTCDHLIKWPDRASLKGKFCSYLAGVCGSSSVVIQNVNAGNKSLDHSEITFRHLAFFCTIYHLHQSDRTDAHSPLVQVKTFPDTGGFVLYRKNADVGIEHKLQHQNDSLSCIPGCSLLSIKSALTLFPSNHSSHVSPAGVMIRVRPTAITSTRFTFSGNATAFGSLTAWLRLLRNTVVSIICLLMWICLVYIHCGIDAGICQRDIRL) contains multiple cysteine residues that may form disulfide bonds affecting structural stability .

How can I design experiments to study trbH protein-protein interactions?

Designing experiments to study trbH protein-protein interactions requires a multi-faceted approach:

• Pull-down assays: Utilize the His-tag on recombinant trbH for pull-down experiments.

  • Immobilize purified His-tagged trbH on Ni-NTA resin

  • Incubate with potential interaction partners from cell lysates

  • Wash extensively to remove non-specific binding proteins

  • Elute bound complexes with imidazole gradient

  • Analyze isolated complexes by SDS-PAGE and mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize trbH protein on a sensor chip via His-tag

  • Flow potential binding partners across the surface

  • Measure real-time binding kinetics

  • Determine association (ka) and dissociation (kd) constants for interactions

  • Calculate binding affinity (KD = kd/ka)

• Bacterial Two-Hybrid System:

  • Create fusion constructs of trbH with one domain of a split reporter protein

  • Create libraries of potential partners fused to the complementary domain

  • Co-express in bacteria and screen for reporter activation

  • Identify and validate positive interactions through secondary assays

• Crosslinking coupled with mass spectrometry:

  • Treat purified trbH with potential partners in the presence of crosslinking agents

  • Digest crosslinked complexes with proteases

  • Analyze by LC-MS/MS to identify crosslinked peptides

  • Use specialized software to map interaction interfaces

This comprehensive approach allows for both identification of novel interaction partners and detailed characterization of binding properties, providing insights into trbH's functional roles within cellular networks.

What cell lines and growth conditions are optimal for high-yield expression of soluble trbH protein?

For high-yield expression of soluble trbH protein, consider the following optimized conditions:

Recommended E. coli strains:

• BL21(DE3): Standard strain for T7 promoter-based expression systems

• Rosetta(DE3): Supplies tRNAs for rare codons that might be present in trbH

• SHuffle T7: Engineered to promote disulfide bond formation in the cytoplasm

Growth media optimization:

• Base medium: 2xYT or Terrific Broth (TB) rather than LB for higher cell density

• Supplementation: 0.5-1% glucose to reduce basal expression before induction

• Trace elements: Addition of ZnSO₄ (0.1 mM) if trbH contains zinc-binding motifs

Growth conditions:

• Initial growth temperature: 37°C until OD₆₀₀ reaches 0.6-0.8

• Post-induction temperature: 16-25°C to reduce inclusion body formation

• Induction strategy: 0.1-0.5 mM IPTG for T7-based systems, or autoinduction media

• Duration: Extended expression (16-24 hours) at lower temperatures

pH-stat fed-batch cultivation:

• Studies with other recombinant proteins show that pH-stat fed-batch cultures can achieve cell concentrations of 27.5 g DCW/liter with co-expression of ftsA and ftsZ genes

• Specific growth rates of approximately 0.13 h⁻¹ can be maintained

• Active production typically begins 16-21 hours after inoculation

This combination of strain selection, media formulation, and growth parameters provides optimal conditions for producing soluble trbH protein with reduced cell filamentation and enhanced volumetric productivity.

How can I address poor solubility issues with recombinant trbH protein?

Poor solubility of recombinant trbH protein can be addressed through multiple complementary approaches:

• Fusion tag optimization:

  • In addition to the His-tag, consider testing alternative solubility-enhancing tags such as MBP (maltose-binding protein), SUMO, or Thioredoxin

  • Implement tag placement studies comparing N-terminal versus C-terminal fusion positions

  • Include TEV or PreScission protease cleavage sites for tag removal after purification

• Expression condition modifications:

  • Reduce expression temperature to 16-20°C after induction

  • Decrease inducer concentration (0.01-0.1 mM IPTG instead of standard 1 mM)

  • Implement slow induction strategies using lactose instead of IPTG

  • Evaluate the effect of co-expressing molecular chaperones like GroEL/GroES

• Buffer optimization for purification:

  • Screen various pH conditions (pH 6.0-9.0) to identify optimal solubility range

  • Test additives including:

    • Glycerol (5-20%)

    • Non-detergent sulfobetaines (NDSB-201, 0.5-1 M)

    • Arginine (0.2-0.5 M)

    • Low concentrations of mild detergents (0.05% Triton X-100)

• Refolding strategies (if inclusion bodies are unavoidable):

  • Solubilize inclusion bodies in 6-8 M urea or 4-6 M guanidine hydrochloride

  • Remove denaturant by step-wise dialysis or rapid dilution

  • Add oxidized/reduced glutathione pairs (1:10 ratio) to facilitate correct disulfide formation

  • Implement artificial chaperone-assisted refolding using cyclodextrins

• Prevent filamentation during high-density cultures:

  • Co-express ftsA and ftsZ genes to maintain normal cell morphology and prevent stress-induced filamentation that occurs during recombinant protein production

  • This approach has been shown to increase both cell growth rate and recombinant protein production in high-density cultures

What analytical methods are most effective for characterizing trbH protein function and structure?

The most effective analytical methods for characterizing trbH protein function and structure include:

This multi-faceted approach provides complementary data on both structure and function, allowing for comprehensive characterization of trbH protein and its potential roles in biological systems.

How should I interpret discrepancies in trbH expression levels between different experimental replicates?

When encountering discrepancies in trbH expression levels between experimental replicates, a systematic analysis approach is essential:

• Categorize variations by magnitude and pattern:

  • Minor variations (<20%): Likely represent normal experimental variance

  • Significant variations (20-50%): May indicate procedural inconsistencies

  • Major variations (>50%): Suggest fundamental technical issues or biological variables

• Analyze potential sources of technical variation:

  • Induction timing: Differences in culture density (OD₆₀₀) at induction point

  • Cell harvest timing: Variations in post-induction harvesting times

  • Expression temperature control: Fluctuations in incubator temperatures

  • Media composition: Batch-to-batch variations in complex media components

• Assess biological factors:

  • Plasmid stability: Loss of expression plasmid without antibiotic pressure

  • Cell viability: Differences in viable cell percentages between cultures

  • Metabolic burden: Variability in cellular stress responses to protein overexpression

  • Cell filamentation: Formation of filamentous cells can affect protein expression; co-expression of ftsA and ftsZ genes may help normalize cell morphology

• Statistical evaluation:

  • Calculate means, standard deviations, and coefficients of variation

  • Perform appropriate statistical tests to determine if differences are significant

  • Use multiple (at least 3) biological replicates and multiple technical replicates

  • Consider power analysis to determine if sample size is sufficient

• Standardization measures for future experiments:

  • Implement stricter controls on induction OD₆₀₀ (±0.05 units)

  • Use single batches of media components for related experiments

  • Maintain master cell banks to reduce genetic drift

  • Monitor growth curves in real-time to ensure consistency

Researchers should note that in studies with other recombinant proteins, co-expression of cell division proteins ftsA and ftsZ reduced variability between cultures by maintaining normal cell morphology and preventing stress-induced filamentation .

What statistical approaches are recommended for analyzing trbH protein interaction data?

When analyzing trbH protein interaction data, the following statistical approaches are recommended for robust interpretation:

• For Surface Plasmon Resonance (SPR) data:

  • Model selection: Use appropriate binding models (1:1, heterogeneous ligand, etc.)

  • Residual analysis: Evaluate systematic deviations from fitted curves

  • Global fitting: Simultaneously fit multiple sensorgrams at different concentrations

  • Statistical tests: Calculate chi-square values to assess goodness of fit

  • Replicate analysis: Perform at least three independent measurements

  • Confidence intervals: Report 95% confidence intervals for ka, kd, and KD values

• For pull-down and co-immunoprecipitation studies:

  • Quantitative western blot analysis: Use standard curves with recombinant standards

  • Normalization strategies: Account for variations in loading and immunoblotting efficiency

  • Statistical significance: Apply t-tests or ANOVA for comparing means across conditions

  • Multiple testing correction: Use Benjamini-Hochberg procedure to control false discovery rate

  • Background subtraction: Implement proper negative controls for non-specific binding

• For high-throughput interaction screening:

  • Scoring functions: Develop normalized scoring systems for interaction strength

  • Threshold determination: Establish statistically justified cutoffs for positive interactions

  • Receiver Operating Characteristic (ROC) analysis: Optimize sensitivity and specificity

  • Machine learning approaches: Implement supervised learning algorithms to classify interactions

  • Network analysis: Apply graph theory metrics to evaluate interaction networks

• For structural data interpretation:

  • Model validation: Use R-factors, Ramachandran plots, and geometric criteria

  • Ensemble analysis: Evaluate precision across multiple models

  • Comparisons between methods: Statistical techniques for integrating data from different structural methods

• General recommendations:

  • Power analysis: Determine appropriate sample sizes beforehand

  • Effect size calculation: Report Cohen's d or similar metrics

  • Bayesian approaches: Consider Bayesian statistics for complex datasets

  • Visualization: Use comprehensive graphical representations of data distributions

This multi-faceted statistical approach ensures robust and reproducible interpretation of protein interaction data while minimizing false positives and negatives.

How can I leverage synthetic biology approaches to study trbH function?

Synthetic biology offers powerful approaches to study trbH function through systematic redesign and engineering:

• Modular domain swapping:

  • Create chimeric proteins by swapping domains between trbH and related proteins

  • Express these chimeras to identify functional modules responsible for specific activities

  • Test complementation of knockout phenotypes with chimeric constructs

• Protein circuit design:

  • Engineer synthetic genetic circuits incorporating trbH

  • Implement feedback loops to study regulation of trbH-dependent processes

  • Create reporter systems that provide real-time visualization of trbH activity

  • Design orthogonal systems to study trbH function in isolation from native cellular processes

• Promoter engineering for controlled expression:

  • Utilize promoter trap systems to discover and optimize promoters for trbH expression

  • Implement dual-promoter systems that have demonstrated increased protein expression in bacterial systems

  • Create inducible promoter systems with varying strengths to titrate trbH concentration

• CRISPR-Cas9 genome editing:

  • Generate precise chromosomal mutations or tags in the native trbH gene

  • Create conditional knockdowns using CRISPR interference (CRISPRi)

  • Implement base editing for single nucleotide modifications without double-strand breaks

• Minimal system reconstitution:

  • Identify the minimal set of components required for trbH function

  • Reconstruct these components in heterologous hosts or cell-free systems

  • Use this minimal system to study functional mechanisms without confounding factors

• High-throughput mutagenesis:

  • Create comprehensive mutant libraries using techniques like deep mutational scanning

  • Implement FACS-based screens to identify functional variants

  • Map the sequence-function relationship of trbH at high resolution

These synthetic biology approaches provide powerful tools for dissecting trbH function beyond traditional biochemical and genetic techniques, offering insights that might be inaccessible through conventional methods.

What are the current limitations in studying trbH and how might they be overcome?

Current limitations in studying trbH protein and potential strategies to overcome them include:

• Limited structural information:

  • Limitation: Absence of high-resolution structural data impedes mechanism understanding

  • Solution: Implement integrative structural biology approaches combining X-ray crystallography, cryo-EM, NMR, and computational modeling to overcome crystallization challenges

• Functional ambiguity:

  • Limitation: Incomplete understanding of physiological roles and functional mechanisms

  • Solution: Combine genetic approaches (knockouts, complementation) with biochemical assays and interactome analysis to elucidate functional networks

• Expression challenges:

  • Limitation: Difficulty obtaining sufficient quantities of properly folded protein

  • Solution: Explore co-expression of ftsA and ftsZ genes to improve cell growth and protein production by preventing stress-induced filamentation

  • Solution: Implement synthetic promoter engineering using promoter trap systems to optimize expression levels

• Membrane association complexities:

  • Limitation: Challenges in studying membrane-associated aspects of trbH function

  • Solution: Develop nanodiscs or liposome-based reconstitution systems specifically optimized for trbH

• Complex formation difficulties:

  • Limitation: Transient or context-dependent interactions might be missed

  • Solution: Implement proximity labeling approaches (BioID, APEX) in physiological settings

• Technology integration:

  • Limitation: Fragmented data from different methodological approaches

  • Solution: Develop integrated data analysis pipelines combining transcriptomics, proteomics, and functional genomics data

• Research coordination:

  • Limitation: Dispersed efforts by different research groups

  • Solution: Establish research consortia focused on trbH and related proteins

This systematic approach to addressing limitations will accelerate progress in understanding trbH structure, function, and biological significance, potentially revealing new applications in biotechnology and medicine.