Recombinant Human KH homology domain-containing protein 1 (KHDC1)

What is KHDC1 and what is known about its molecular function?

KHDC1 (KH Homology Domain Containing 1) is a human protein containing a KH domain, which is typically associated with RNA binding capabilities. The KH domain is characterized by its ability to recognize and bind specific RNA sequences . While the exact molecular function of KHDC1 remains under investigation, its structural features suggest involvement in post-transcriptional regulation processes, potentially including RNA stability control or translational regulation.

The full-length human KHDC1 protein consists of 237 amino acids with a molecular weight of approximately 18.3 kDa . The presence of the KH domain indicates potential roles in mRNA processing, localization, or regulatory pathways. Researchers should note that KHDC1 has a paralog called KHDC1L, which shares significant sequence homology but may have distinct functions .

How should recombinant KHDC1 be properly stored and handled?

For optimal stability, recombinant KHDC1 should be stored at -80°C in an appropriate buffer system. Based on established protocols, a suitable storage buffer contains 25 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, and 1% Sarkosyl . This formulation helps maintain protein stability during freeze-thaw cycles.

When working with the protein:

• Limit freeze-thaw cycles to 2-3 times to prevent degradation

• Thaw the protein on ice before use

• Consider aliquoting into single-use tubes upon initial thawing

• For long-term storage, addition of carrier proteins (0.1% HSA or BSA) may enhance stability

What expression systems yield optimal results for recombinant KHDC1 production?

Escherichia coli Expression System:

E. coli represents the most commonly utilized expression system for recombinant KHDC1 production . This bacterial system offers several advantages for KHDC1 expression:

While E. coli is effective for basic structural studies, researchers investigating post-translational modifications or complex folding requirements may need to consider mammalian expression systems like HEK-293 cells, which can provide more native-like protein modifications .

What purification strategies are most effective for recombinant His-tagged KHDC1?

A multi-step purification process is recommended to achieve high purity (>80-90%) recombinant KHDC1:

• Immobilized Metal Affinity Chromatography (IMAC):

  • Use Ni-NTA or Co-based resins

  • Equilibrate with buffer containing 25 mM Tris-HCl (pH 8.0), 150-300 mM NaCl, 10-20 mM imidazole

  • Apply clarified lysate

  • Wash with increasing imidazole concentrations (20-50 mM)

  • Elute with high imidazole (250-500 mM)

• Size Exclusion Chromatography (SEC):

  • Further purify IMAC-purified protein

  • Remove aggregates and improve homogeneity

  • Recommended buffer: 25 mM Tris-HCl (pH 8.0), 150 mM NaCl

• Quality Control:

  • Verify purity by SDS-PAGE (>80-90% purity is typically achievable)

  • Confirm identity via Western blot with anti-His or anti-KHDC1 antibodies

  • Assess homogeneity by analytical SEC

The addition of detergents like Sarkosyl (1%) may improve solubility during purification . For applications requiring higher purity, ion exchange chromatography can be incorporated as an intermediate step.

How does the His-tag affect KHDC1 structure and function, and when should it be removed?

The N-terminal His-tag (typically 6× histidine residues) can potentially influence KHDC1's structure and function in several ways:

Potential Impacts:

• May alter protein folding or oligomerization state

• Could affect binding to physiological partners

• Might introduce artifact interactions with negatively charged molecules

• Potential interference with N-terminal functional domains

Tag Removal Considerations:
For studies investigating native KHDC1 function, particularly those examining protein-protein or protein-RNA interactions, His-tag removal should be considered. This can be accomplished by incorporating a protease cleavage site (TEV, PreScission, or thrombin) between the tag and the protein .

What are the critical considerations for designing experiments to study KHDC1-RNA interactions?

When investigating KHDC1-RNA interactions, researchers should consider:

• RNA Binding Assay Selection:

  • RNA Electrophoretic Mobility Shift Assay (EMSA) for qualitative binding analysis

  • Filter binding assays for quantitative Kd determination

  • CLIP-seq (Cross-linking immunoprecipitation) for in vivo RNA targets identification

  • Surface Plasmon Resonance for real-time binding kinetics

• RNA Target Selection:

  • Begin with known KH-domain binding motifs (typically C/U-rich sequences)

  • Include both specific and non-specific RNA controls

  • Consider both short oligonucleotides and structured RNAs

• Buffer Optimization:

  • Test multiple pH conditions (typically pH 7.0-8.0)

  • Optimize salt concentration (50-150 mM NaCl)

  • Include Mg²⁺ (1-5 mM) for RNA structure stabilization

  • Add RNase inhibitors to prevent degradation

• Binding Conditions:

  • Temperature effects (4°C vs. room temperature vs. 37°C)

  • Incubation time optimization (15 min to 1 hour)

  • Protein:RNA ratio titration

The recombinant nature of the KHDC1 protein used may influence binding properties, particularly if post-translational modifications present in vivo are absent in the E. coli-expressed version . Researchers should validate key findings using mammalian-expressed KHDC1 when possible.

How can researchers differentiate between KHDC1 and its paralog KHDC1L in experimental systems?

KHDC1 and KHDC1L share significant sequence homology but differ in several key aspects that can be leveraged for experimental differentiation:

Experimental Approaches for Differentiation:

• Antibody-based Methods:

  • Generate peptide antibodies against unique regions not conserved between the paralogs

  • Validate antibody specificity using recombinant proteins of both paralogs

  • Employ immunoprecipitation followed by mass spectrometry for definitive identification

• Nucleic Acid-based Methods:

  • Design paralog-specific PCR primers targeting divergent regions

  • Employ paralog-specific siRNAs for selective knockdown experiments

  • Use CRISPR-Cas9 with guides designed to unique genomic regions

• Expression Analysis:

  • Compare tissue distribution patterns

  • Analyze subcellular localization differences

When conducting functional studies, researchers should confirm which paralog they are studying, as the function of KHDC1L may be distinct from KHDC1 despite their structural similarities .

What are common challenges in recombinant KHDC1 expression and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant KHDC1:

Challenge 1: Low Expression Yield

• Solution: Optimize codon usage for E. coli by using codon-optimized synthetic genes

• Solution: Test different E. coli strains (BL21, Rosetta, Arctic Express)

• Solution: Adjust induction conditions (IPTG concentration, temperature, duration)

Challenge 2: Poor Solubility/Inclusion Body Formation

• Solution: Reduce induction temperature (16-20°C)

• Solution: Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

• Solution: Use solubility-enhancing fusion partners (SUMO, MBP, GST)

• Solution: Add solubilizing agents to lysis buffer (1% Sarkosyl has proven effective)

Challenge 3: Protein Degradation

• Solution: Add protease inhibitors during purification

• Solution: Use protease-deficient E. coli strains

• Solution: Maintain cold temperatures throughout purification

• Solution: Minimize purification time

Challenge 4: Protein Aggregation

• Solution: Include 10% glycerol in buffers

• Solution: Add reducing agents (DTT or β-mercaptoethanol)

• Solution: Optimize salt concentration (150-300 mM NaCl)

• Solution: Filter protein solutions before storage

How can researchers verify the structural integrity and activity of purified recombinant KHDC1?

Multiple complementary approaches should be employed to ensure recombinant KHDC1 is properly folded and functionally active:

• Structural Integrity Assessment:

  • Circular Dichroism (CD) spectroscopy to analyze secondary structure content

  • Thermal shift assays to evaluate protein stability

  • Dynamic Light Scattering to assess monodispersity

  • Limited proteolysis to probe for well-folded domains resistant to digestion

• Functional Validation:

  • RNA binding assays using known KH domain ligands

  • Co-immunoprecipitation with known interaction partners

  • Activity assays specific to hypothesized function

• Quality Control Metrics:

  • Purity assessment by SDS-PAGE (should exceed 80-90%)

  • Western blotting to confirm identity

  • Mass spectrometry to verify sequence and identify potential modifications

  • Endotoxin testing for applications in cell culture or in vivo studies

Researchers should establish positive controls where possible, such as commercially available recombinant KHDC1 with verified activity, to benchmark their preparation against known standards .

What are emerging techniques for studying KHDC1 function in cellular contexts?

Several cutting-edge methodologies are advancing our understanding of KHDC1 biology:

• Proximity Labeling Technologies:

  • BioID or TurboID fusion constructs to identify proximal proteins in living cells

  • APEX2 fusion proteins for spatially-restricted proteomics

  • These approaches can reveal the KHDC1 interactome in different cellular compartments

• Advanced Imaging Approaches:

  • Live-cell imaging with fluorescently-tagged KHDC1 to track dynamics

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization

  • FRET-based biosensors to detect KHDC1 interactions in real-time

• Genomic Engineering:

  • CRISPR-Cas9 knock-in of endogenously tagged KHDC1

  • CRISPR activation or interference to modulate KHDC1 expression

  • CRISPR base editing for studying specific amino acid contributions

• Systems Biology Integration:

  • Multi-omics approaches combining proteomics, transcriptomics, and metabolomics

  • Network analysis to position KHDC1 in regulatory pathways

  • Machine learning prediction of KHDC1 functions based on similar domains

These methodologies provide complementary insights when combined with biochemical studies using recombinant KHDC1 protein , allowing researchers to bridge in vitro observations with cellular functions.

How can differences between recombinant and native KHDC1 impact experimental interpretation?

When working with recombinant KHDC1, researchers should recognize several key differences from the native protein that may influence experimental results:

1. Post-translational Modifications (PTMs):

• Recombinant KHDC1 from E. coli lacks eukaryotic PTMs (phosphorylation, methylation, etc.)

• These modifications may alter binding properties, localization, or stability

• Solution: Consider mammalian expression systems for PTM-dependent studies

• Solution: Complement E. coli-derived protein studies with cell-based validation

2. Structural Considerations:

• Presence of tags (particularly His-tags) may affect structure and function

• Recombinant protein may adopt different conformations due to folding environment

• Solution: Compare tagged and untagged versions in critical experiments

• Solution: Use structural validation methods (CD spectroscopy, thermal shift assays)

3. Binding Partner Availability:

• In vitro systems lack cellular cofactors that may be required for native function

• Protein complex formation may be essential for proper KHDC1 activity

• Solution: Include relevant cofactors in reconstitution experiments

• Solution: Consider pull-down experiments to isolate natural complexes

4. Concentration Effects:

• Recombinant protein experiments often use non-physiological concentrations

• High concentrations may promote non-specific interactions

• Solution: Perform concentration-dependent experiments to identify specific effects

• Solution: Validate with cellular studies using endogenous levels

By carefully considering these differences, researchers can design experiments that appropriately bridge the gap between recombinant protein studies and physiological KHDC1 function.