Recombinant Escherichia coli Uncharacterized protein ymfR (ymfR)

What is ymfR and where is it found in E. coli?

YmfR is an uncharacterized protein encoded by the ymfR gene (b1150) in Escherichia coli K-12. It is a relatively small protein of 60 amino acids associated with the e14 prophage region of the E. coli genome. The protein belongs to the phage terminase protein A family and is considered phage or prophage-related in function . The protein has been identified as part of the E. coli genome but its precise biological role remains to be fully elucidated through ongoing research.

What is the amino acid sequence and basic properties of ymfR protein?

The full-length ymfR protein consists of 60 amino acids with the sequence: MIMLILAPLVGVLGALLLAYGAWLIYPPAGFVVAGALCLFWSWLVARYLDRTQSSVGGGK . Based on this sequence, ymfR appears to have hydrophobic regions consistent with a membrane-associated protein. The multiple hydrophobic stretches suggest it may be integrated into bacterial membranes, potentially as part of phage-related functions. When expressing this protein recombinantly, researchers should consider these properties when designing solubilization and purification strategies.

How does ymfR relate to other proteins in the E. coli proteome?

YmfR is part of an interaction network primarily involving other proteins encoded by the e14 prophage. String database analysis reveals strong predicted functional partnerships with several proteins including:

This network suggests ymfR may function as part of the e14 prophage regulatory system, potentially involved in phage lifecycle regulation or host-phage interactions .

What are the optimal conditions for recombinant expression of ymfR in E. coli?

For recombinant expression of ymfR, researchers typically use E. coli expression systems with the full-length protein (amino acids 1-60) fused to an N-terminal His tag . The optimal expression conditions should consider:

• Selection of expression vector: A T7 promoter-based system like pET vectors offers tight regulation and high expression levels when induced .

• E. coli strain selection: BL21(DE3) derivatives are commonly used for membrane-associated proteins, but strains with modified reduction potential like Origami might improve folding .

• Induction parameters: IPTG concentration (typically 0.1-1.0 mM), temperature (reduced to 16-25°C for membrane proteins), and induction time (4-16 hours) should be optimized.

• Media composition: Rich media like LB for high biomass or defined media for isotope labeling in structural studies.

Given ymfR's hydrophobic nature, expression conditions may need to be modified to prevent inclusion body formation or protein misfolding.

What purification strategies work best for recombinant ymfR protein?

Purification of recombinant His-tagged ymfR typically involves:

• Cell lysis: For membrane-associated proteins like ymfR, detergent-based lysis buffers (containing mild detergents like n-dodecyl β-D-maltoside or Triton X-100) may improve solubilization.

• Affinity chromatography: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is the primary purification step for His-tagged ymfR .

• Buffer optimization:

  • Lysis buffer: Tris/PBS-based buffer, pH 8.0

  • Elution buffer: Imidazole gradient (20-250 mM)

  • Storage buffer: Tris/PBS-based buffer with 6% trehalose, pH 8.0

• Additional purification: Size exclusion chromatography can be used to achieve >90% purity, removing aggregates and contaminants.

• Storage conditions: Store lyophilized protein at -20°C/-80°C. After reconstitution, add 5-50% glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C to avoid repeated freeze-thaw cycles .

How can researchers improve the solubility of recombinant ymfR during expression?

Improving solubility of hydrophobic proteins like ymfR requires strategic approaches:

• Fusion partners: Beyond the His-tag, larger solubility-enhancing fusion partners like maltose-binding protein (MBP), glutathione S-transferase (GST), or SUMO can significantly improve solubility .

• Expression temperature: Lowering expression temperature to 16-20°C slows protein production and may improve folding of membrane-associated proteins.

• Co-expression strategies: Co-expressing chaperones like GroEL/GroES or specialized membrane protein chaperones can improve proper folding.

• Detergent screening: For membrane proteins, early addition of mild detergents during cell lysis can improve extraction and prevent aggregation.

• Refolding protocols: If ymfR forms inclusion bodies despite optimization, denaturation with urea or guanidinium hydrochloride followed by controlled refolding may be necessary.

Recent research has shown that even traditionally difficult-to-express proteins can achieve higher solubility in BL21(DE3) strains when fusion partners like GFP are employed .

What computational tools can predict the structure and function of ymfR?

In the absence of experimental structural data for ymfR, computational approaches provide valuable insights:

• Sequence homology analysis: Tools like BLAST and HHpred can identify distant homologs with known functions.

• Secondary structure prediction: PSIPRED, JPred, or SOPMA can predict α-helices, β-sheets, and transmembrane regions in ymfR.

• Tertiary structure prediction: AlphaFold2 and RoseTTAFold have revolutionized protein structure prediction for uncharacterized proteins.

• Function prediction: InterProScan can identify functional domains, while tools like ProFunc and COFACTOR predict potential biochemical functions.

• Protein-protein interaction prediction: STRING database already shows ymfR's interaction network with other e14 prophage proteins, providing functional context .

For ymfR specifically, these tools might predict transmembrane regions consistent with its potential role in phage assembly or host cell membrane interaction during the phage lifecycle.

How can researchers experimentally determine the structure of ymfR?

Experimental structure determination for ymfR could employ:

• X-ray crystallography: Challenging for membrane proteins, but possible with:

  • Detergent screening to find optimal solubilization conditions

  • Lipidic cubic phase crystallization techniques

  • Fusion to crystallization chaperones like T4 lysozyme

• NMR spectroscopy: Suitable for small proteins like ymfR (60 aa):

  • Requires isotope labeling (15N, 13C) during expression

  • May require specialized membrane-mimetic environments (micelles, bicelles)

  • Can provide dynamic information about protein flexibility

• Cryo-electron microscopy: If ymfR forms part of a larger complex:

  • May be combined with cross-linking studies to capture interactions

  • Single-particle analysis if sufficient size (often combined with fusion partners)

• Circular dichroism spectroscopy: For secondary structure content analysis:

  • Provides percentages of α-helix, β-sheet, and random coil

  • Can monitor structural changes under varying conditions

Given ymfR's small size and potential membrane association, NMR may be the most promising approach for high-resolution structural studies.

What experimental approaches can determine ymfR's function in E. coli?

Determining the function of uncharacterized proteins like ymfR requires multiple complementary approaches:

• Gene knockout/complementation studies:

  • CRISPR-Cas9 or λ-Red recombineering to generate ymfR deletion strains

  • Phenotypic analysis under various growth conditions

  • Complementation with wild-type or mutant ymfR

• Protein-protein interaction studies:

  • Pull-down assays using His-tagged ymfR

  • Bacterial two-hybrid screening

  • Co-immunoprecipitation with predicted partners like stfP, ymfM, or ymfQ

  • Cross-linking mass spectrometry (XL-MS) to capture transient interactions

• Localization studies:

  • Fluorescent protein fusions to determine subcellular localization

  • Fractionation experiments to determine membrane association

  • Immunogold electron microscopy for precise localization

• Transcriptomic/proteomic analysis:

  • RNA-seq comparing wild-type and ymfR deletion strains

  • Proteome analysis to identify regulated proteins

  • Metabolomic analysis to identify affected pathways

Given ymfR's association with prophage e14 proteins, experiments should particularly focus on phage-related functions like DNA packaging, virion assembly, or host cell lysis regulation.

How should researchers design experiments to investigate ymfR's role in phage biology?

To investigate ymfR's role in phage biology:

• Prophage induction experiments:

  • Treat E. coli containing e14 prophage with DNA-damaging agents (UV, mitomycin C)

  • Compare phage production in wild-type vs. ymfR deletion strains

  • Quantify phage particles by plaque assays or qPCR

• Phage lifecycle stage determination:

  • Temporal expression analysis of ymfR during prophage induction

  • Co-localization with other phage proteins during assembly

  • Electron microscopy to visualize potential structural roles

• Host-phage interaction studies:

  • Bacterial membrane integrity assays in presence/absence of ymfR

  • Liposome binding/disruption assays using purified ymfR

  • Phage DNA packaging assays if ymfR functions in terminase complex

• Site-directed mutagenesis:

  • Create point mutations in conserved residues of ymfR

  • Assess effects on phage production and e14 functions

  • Examine protein-protein interactions with predicted partners

This experimental framework follows similar approaches used to characterize other initially uncharacterized proteins in E. coli, as described in studies of transcription factors .

How can ymfR research benefit from current high-throughput technologies?

High-throughput technologies offer powerful approaches for ymfR characterization:

• ChIP-exo analysis: If ymfR has DNA-binding properties like some prophage proteins, this technique can identify genomic binding sites with single-nucleotide resolution .

• Transposon insertion sequencing (Tn-seq):

  • Identify genetic interactions by creating a transposon library in ymfR+ and ymfR- backgrounds

  • Compare fitness effects to identify synthetic lethal/sick interactions

• Barcode-sequencing screens:

  • Test thousands of conditions simultaneously to identify when ymfR becomes essential

  • Screen chemical libraries for compounds that specifically affect ymfR function

• High-throughput protein-protein interaction screening:

  • Protein microarrays to identify interaction partners beyond known e14 proteins

  • Split reporter assays (luciferase, GFP) in arrayed format

• Cryo-electron tomography:

  • Visualize structural changes in phage assembly or membrane structures in ymfR mutants

  • Correlative light and electron microscopy to capture dynamic processes

These approaches align with methods successfully used to characterize previously uncharacterized transcription factors in E. coli, as described in recent literature .

What controls should be included when studying recombinant ymfR function?

Rigorous controls are essential for reliable ymfR functional studies:

• Expression controls:

  • Empty vector controls to account for expression system effects

  • Non-related protein expressed under identical conditions

  • Untagged ymfR to control for tag interference with function

• Functional assay controls:

  • Known e14 prophage proteins with established functions

  • Inactive mutants of ymfR (e.g., site-directed mutations)

  • Dose-response relationships to establish specificity

• Specificity controls:

  • Homologous proteins from related phages/prophages

  • Scrambled or randomized protein sequences of similar composition

  • Competition assays with unlabeled protein

• Technical controls:

  • Multiple biological and technical replicates

  • Alternative methods to confirm key findings

  • Blinding of samples during analysis when possible

These controls help distinguish true biological functions from artifacts, particularly important for uncharacterized proteins where biases about function are minimal.

What are common challenges in expressing and purifying recombinant ymfR?

Common challenges with ymfR expression and purification include:

• Low expression yields:

  • Problem: Hydrophobic proteins often express poorly

  • Solution: Try specialized strains like C41/C43(DE3) designed for membrane proteins, or codon-optimized constructs

• Inclusion body formation:

  • Problem: Aggregation due to hydrophobicity

  • Solution: Lower induction temperature (16°C), reduce IPTG concentration, or use solubility-enhancing fusion tags like MBP or SUMO

• Protein degradation:

  • Problem: Small proteins can be rapidly degraded

  • Solution: Add protease inhibitors, express in protease-deficient strains, optimize lysis conditions

• Purification difficulties:

  • Problem: Non-specific binding to purification resins

  • Solution: Optimize imidazole concentrations in binding/wash buffers, add low concentrations of detergents

• Loss of activity after purification:

  • Problem: Protein misfolding or denaturation during purification

  • Solution: Include stabilizing agents like trehalose (as mentioned in the product specifications) , optimize buffer composition

Addressing these challenges requires systematic optimization of expression and purification conditions.

How can researchers troubleshoot inconsistent results in ymfR functional assays?

When facing inconsistent results in ymfR functional assays:

• Protein quality assessment:

  • Verify protein integrity by SDS-PAGE before each experiment

  • Check for degradation products using Western blotting

  • Assess protein folding using circular dichroism or fluorescence spectroscopy

• Assay standardization:

  • Develop quantitative assays with internal standards

  • Establish dose-response relationships

  • Ensure consistent reaction conditions (temperature, pH, ionic strength)

• Batch variation analysis:

  • Track protein production batches and correlate with assay performance

  • Implement quality control metrics for each preparation

  • Consider single-batch studies for critical experiments

• Environmental variables:

  • Control for media composition effects on host cells

  • Account for growth phase differences in bacterial cultures

  • Monitor temperature fluctuations during assays

• Data analysis refinement:

  • Apply appropriate statistical tests for biological variability

  • Consider outlier identification and handling protocols

  • Implement blinded analysis where feasible

Methodical troubleshooting approaches similar to those used in characterizing other uncharacterized E. coli proteins can help address these challenges .

What strategies help overcome difficulties in determining the function of uncharacterized proteins like ymfR?

Strategies to determine functions of uncharacterized proteins like ymfR include:

• Comparative genomics approaches:

  • Examine genomic context across related bacteria

  • Identify co-occurrence patterns with genes of known function

  • Analyze synteny and conservation in phage genomes

• Phenotypic microarrays:

  • Test growth under hundreds of conditions to identify specific sensitivities in ymfR mutants

  • Look for chemical or environmental stresses that differentiate wild-type from mutant

• Physiological perturbation:

  • Challenge cells with various stresses (oxidative, osmotic, pH, antibiotics)

  • Monitor fitness differences between wild-type and ymfR mutant strains

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Use network analysis to position ymfR in biological pathways

  • Apply machine learning to predict function from integrated datasets

• Heterologous expression:

  • Express ymfR in different bacterial hosts lacking e14 prophage

  • Look for gain-of-function phenotypes that suggest function

These approaches parallel successful methods used to characterize other uncharacterized proteins in E. coli, such as the work on novel transcription factors described in the literature .

How might ymfR research contribute to understanding prophage-host interactions?

Research on ymfR could advance understanding of prophage-host interactions through:

• Prophage maintenance mechanisms:

  • Investigate if ymfR contributes to prophage stability within the host genome

  • Determine if ymfR affects prophage induction rates under stress conditions

  • Examine potential roles in prophage excision regulation

• Host fitness contributions:

  • Assess how ymfR affects bacterial growth under various conditions

  • Determine if ymfR confers resistance to external phage infection (superinfection exclusion)

  • Investigate potential metabolic effects on host cells

• Phage-host co-evolution:

  • Compare ymfR sequences across E. coli strains with e14 prophages

  • Identify selection pressures through comparative sequence analysis

  • Investigate horizontal gene transfer patterns involving ymfR

• Phage-mediated virulence:

  • Examine if ymfR affects expression of host virulence factors

  • Investigate potential roles in bacterial stress responses relevant to pathogenesis

  • Determine if ymfR influences biofilm formation or other community behaviors

This research direction is particularly relevant given the growing recognition of prophages as important modulators of bacterial physiology and virulence.

What are the implications of ymfR research for synthetic biology applications?

YmfR research could contribute to synthetic biology in several ways:

• Phage-based genetic tools:

  • If ymfR functions in DNA packaging or virion assembly, it could be engineered for improved phage display technologies

  • Development of controlled gene delivery systems based on modified phage components

• Membrane-targeting applications:

  • If ymfR interacts with bacterial membranes, engineered variants could create selective membrane permeabilization systems

  • Development of targeted antimicrobial peptides based on ymfR structure

• Regulatory element development:

  • YmfR's interaction network could be repurposed to create novel genetic switches

  • Integration into synthetic gene circuits for programmed bacterial behavior

• Biosensing applications:

  • If ymfR responds to specific environmental signals, it could be engineered as a biosensor component

  • Development of whole-cell biosensors for detecting conditions that trigger prophage induction

These applications would build upon the expanding toolkit of bacterial components being repurposed for synthetic biology applications.

How can researchers integrate ymfR studies with broader research on uncharacterized bacterial proteins?

Integration of ymfR research with broader studies on uncharacterized proteins requires:

• Standardized characterization pipelines:

  • Apply consistent experimental workflows like those described for transcription factor characterization

  • Contribute to community resources and databases for uncharacterized proteins

  • Implement FAIR (Findable, Accessible, Interoperable, Reusable) data principles

• Collaborative research networks:

  • Participate in consortium efforts focused on systematic protein characterization

  • Share reagents, protocols, and negative results to accelerate research

  • Engage with structural genomics initiatives for high-throughput structure determination

• Machine learning integration:

  • Contribute data to improve prediction algorithms for protein function

  • Apply emerging AI tools to analyze ymfR properties in context of other proteins

  • Develop new computational approaches to connect sequence, structure, and function

• Funding opportunities alignment:

  • Structure research to align with initiatives like Google Academic Research Awards

  • Develop grant proposals targeting NIH research training programs

  • Focus on interdisciplinary approaches that connect multiple research domains

This integration ensures that insights from ymfR research contribute to broader understanding of uncharacterized proteins, which remain a significant portion of bacterial proteomes.