LAG-1 Human, His

What is LAG-1 and what are its human homologs?

LAG-1 (Lin-12 And Glp-1 phenotype 1) is a transcriptional regulator originally identified in Caenorhabditis elegans as a central component in LIN-12 and GLP-1 mediated signal transduction. The human homolog of LAG-1 is C Promoter Binding Factor 1 (CBF1), also known as Recombination Signal Binding Protein For Immunoglobulin Kappa J Region (RBPJ). This protein functions as a key transcriptional regulator in the Notch signaling pathway . The LAG-1 protein exhibits specific DNA-binding activity, recognizing the consensus sequence RTGGGAA, which is conserved across species from nematodes to humans .

What functional domains characterize the LAG-1/CBF1 protein structure?

The LAG-1/CBF1 protein contains several conserved domains that contribute to its function as a DNA-binding transcriptional regulator. These domains include:

• An N-terminal domain involved in protein-protein interactions

• A central DNA-binding domain that recognizes the RTGGGAA motif

• A C-terminal domain that interacts with co-repressors and co-activators

These structural features enable LAG-1/CBF1 to function as a molecular switch, transitioning between transcriptional repression and activation depending on its interaction partners .

Why are His-tagged versions of LAG-1/CBF1 important for research?

His-tagged versions of LAG-1/CBF1 provide researchers with a valuable tool for protein purification, detection, and functional studies. The histidine tag, typically consisting of 6-10 consecutive histidine residues, offers several advantages:

• Enables efficient single-step purification using immobilized metal affinity chromatography (IMAC)

• Facilitates protein detection via anti-His antibodies

• Can be used for pull-down assays to identify interaction partners

• Allows for consistent protein yield and purity ≥90% when properly optimized

For optimal results, researchers typically add the His-tag to either the N- or C-terminus of the protein, with careful consideration of potential interference with protein folding or function.

How do LAG-1/CBF1 binding patterns differ between normal and disease states?

In normal cellular contexts, LAG-1/CBF1 binds to specific DNA sequences (RTGGGAA) within the promoters and enhancers of Notch pathway target genes. This binding pattern is tightly regulated and context-dependent. In disease states, particularly in cancers with dysregulated Notch signaling, LAG-1/CBF1 binding patterns may be altered in several ways:

• Aberrant recruitment to non-canonical target sites

• Altered binding dynamics due to mutations in the DNA-binding domain

• Changed genomic distribution due to chromatin accessibility modifications

• Disrupted interaction with co-factors affecting binding stability

Research using ChIP-seq with His-tagged LAG-1/CBF1 can provide genome-wide binding profiles to compare normal versus disease states, offering insights into pathological mechanisms and potential therapeutic targets.

What experimental approaches best characterize LAG-1/CBF1 interactions with chromatin remodeling complexes?

Characterizing LAG-1/CBF1 interactions with chromatin remodeling complexes requires a multi-faceted approach:

• Proximity labeling technologies: BioID or APEX2 fused to His-tagged LAG-1/CBF1 to identify proteins in close proximity

• Sequential ChIP (ChIP-reChIP): To determine co-occupancy of LAG-1/CBF1 and specific chromatin remodelers at genomic loci

• Mass spectrometry: Following immunoprecipitation with anti-His antibodies to identify LAG-1/CBF1-associated proteins

• FRET/BRET assays: To study dynamic interactions in living cells

• CUT&RUN or CUT&Tag: For high-resolution mapping of LAG-1/CBF1 and chromatin remodeler co-localization

These approaches can reveal how LAG-1/CBF1 coordinates with chromatin remodeling complexes to regulate gene expression in different cellular contexts.

How do post-translational modifications affect LAG-1/CBF1 function?

LAG-1/CBF1 undergoes various post-translational modifications (PTMs) that modulate its activity:

His-tagged LAG-1/CBF1 provides an excellent tool for enriching the protein for PTM analysis through affinity purification followed by mass spectrometry or western blotting with modification-specific antibodies. Understanding these modifications is crucial for developing targeted therapeutic approaches.

What are the optimal conditions for expressing and purifying His-tagged human LAG-1/CBF1?

Optimal expression and purification of His-tagged human LAG-1/CBF1 requires careful optimization:

Expression system recommendations:

• E. coli BL21(DE3) for the DNA-binding domain alone

• Insect cells (Sf9 or High Five) for full-length protein with proper folding

• Mammalian expression (HEK293 or CHO cells) for studying post-translational modifications

Purification protocol:

• Lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• IMAC purification using Ni-NTA or Co2+ resins

• Imidazole gradient elution (50-250 mM)

• Size exclusion chromatography to remove aggregates

• Storage in buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol

For highest purity (≥90%), additional purification steps such as ion exchange chromatography may be necessary . Aliquoting into small volumes and flash freezing is recommended for long-term storage to maintain protein activity.

How can DNA-binding characteristics of LAG-1/CBF1 be accurately assessed?

Several complementary approaches can be employed to characterize LAG-1/CBF1 DNA binding:

• Electrophoretic Mobility Shift Assay (EMSA): Provides qualitative assessment of binding to the RTGGGAA motif and variants

• Surface Plasmon Resonance (SPR): Enables measurement of binding kinetics and affinity constants

• Microscale Thermophoresis (MST): Allows determination of binding constants in solution

• ChIP-seq with His-tag antibodies: Identifies genome-wide binding sites

• DNA footprinting: Determines precise nucleotides contacted by the protein

When studying the DNA-binding properties of LAG-1/CBF1, it's crucial to consider that the protein binds specifically to the RTGGGAA sequence motif . Experimental design should include positive controls with known binding sites and negative controls with mutated sequences.

What strategies exist for studying LAG-1/CBF1 in the context of Notch signaling complexes?

To study LAG-1/CBF1 within Notch signaling complexes:

• Co-immunoprecipitation using His-tag: Pull down His-tagged LAG-1/CBF1 and identify associated proteins

• Proximity labeling: Fuse BioID or APEX2 to LAG-1/CBF1 to identify nearby proteins

• FRET/BRET analysis: Measure direct protein-protein interactions in living cells

• Single-molecule imaging: Visualize complex assembly and dynamics in real-time

• Cryo-EM: Determine structural organization of LAG-1/CBF1-containing complexes

A combined approach using these methodologies can provide comprehensive understanding of how LAG-1/CBF1 functions within larger transcriptional complexes. For instance, integrating ChIP-seq data with proteomic analyses can reveal how different complex compositions affect genomic targeting.

How can specificity issues with His-tagged LAG-1/CBF1 be addressed?

Researchers working with His-tagged LAG-1/CBF1 may encounter specificity challenges:

Common problems and solutions:

• Non-specific binding during purification

  • Increase imidazole concentration in wash buffers (20-50 mM)

  • Add low concentrations of non-ionic detergents (0.01-0.05% Tween-20)

  • Use cobalt resins instead of nickel for higher specificity

• Tag interference with protein function

  • Test both N-terminal and C-terminal tag positions

  • Include a flexible linker sequence between the tag and protein

  • Consider TEV protease cleavage sites for tag removal after purification

• Cross-reactivity in immunoprecipitation experiments

  • Perform stringent pre-clearing steps

  • Include competitor proteins in binding buffers

  • Validate with alternative tagging systems (FLAG, Strep) for confirmation

Using appropriate controls and optimization can significantly improve specificity when working with His-tagged LAG-1/CBF1 proteins.

What approaches can resolve contradictory data regarding LAG-1/CBF1 binding partners?

When faced with contradictory data about LAG-1/CBF1 interactions:

• Employ orthogonal detection methods:

  • Compare results from different techniques (co-IP, yeast two-hybrid, proximity labeling)

  • Validate interactions in multiple cell types and under different conditions

• Consider context-dependency:

  • Activation state of Notch signaling may affect interactions

  • Cell type-specific cofactors may modulate binding patterns

  • Post-translational modifications can alter interaction profiles

• Analyze interaction kinetics:

  • Some interactions may be transient or weaker than others

  • Time-resolved experiments can capture dynamic interaction changes

• Examine subcellular localization:

  • Co-localization studies can confirm spatial proximity

  • Fractionation experiments can identify compartment-specific interactions

• Use domain mapping:

  • Identify specific domains mediating each interaction

  • Create domain-specific mutants to validate functional importance

How is LAG-1/CBF1 being targeted in therapeutic development?

Current therapeutic strategies targeting LAG-1/CBF1 include:

• Small molecule inhibitors of LAG-1/CBF1-DNA binding

• Peptide mimetics that disrupt protein-protein interactions

• Stapled peptides targeting the Notch-LAG-1/CBF1 interface

• Bifunctional degraders using PROTAC technology

• Gene editing approaches to modulate LAG-1/CBF1 expression

His-tagged LAG-1/CBF1 is particularly valuable in drug screening assays, allowing for high-throughput identification of compounds that disrupt specific interactions. Structural studies of His-tagged LAG-1/CBF1 bound to DNA or protein partners provide crucial insights for rational drug design approaches.

How do computational models integrate LAG-1/CBF1 activity in system-wide analyses?

Computational approaches for studying LAG-1/CBF1 include:

• Network modeling: Integration of LAG-1/CBF1 into larger Notch signaling networks

• Machine learning algorithms: Prediction of LAG-1/CBF1 binding sites from genomic data

• Molecular dynamics simulations: Analysis of LAG-1/CBF1 conformational changes upon binding

• Multi-omics data integration: Combining ChIP-seq, RNA-seq, and proteomics data

Similar to the LAG-1 model described in search result , these computational approaches can capture complex interactions between different components of the signaling pathway across multiple timescales, from fast molecular interactions to slower regulatory responses .