Recombinant Human HHIP-like protein 2 (HHIPL2)

Basic Research Questions

• What is HHIPL2 and what are its basic functional characteristics?

  HHIPL2 (HHIP-like protein 2) is a human protein encoded by the HHIPL2 gene (Gene ID: 79802) located on chromosome 1. It has several alternate names including HHIP3, KIAA1822L, and UNQ841/PRO1779 . According to UniProt (ID: Q6UWX4), HHIPL2 is predicted to enable catalytic activity and is primarily located in the extracellular region . While its precise functions remain under investigation, its classification suggests possible involvement in developmental processes. The protein exhibits high sequence conservation across mammalian species, with approximately 85% sequence identity between human and mouse/rat orthologs .

• What expression systems are recommended for producing recombinant HHIPL2?

  The primary documented expression system for recombinant human HHIPL2 is glycoengineered Pichia pastoris . This system has been specifically selected because:

  Expression System	Advantages for HHIPL2 Production	Considerations
  Glycoengineered P. pastoris	- Produces human-like post-translational modifications - Efficient secretion to medium - Cost-effective compared to mammalian systems - Scalable production capacity	- Requires optimization of growth conditions - Induction parameters need careful monitoring
  E. coli	- Rapid expression - High yield potential	- Lacks glycosylation machinery - May form inclusion bodies with complex proteins
  Mammalian cells	- Native-like glycosylation patterns - Proper protein folding	- Higher production cost - Longer production times

  For HHIPL2 specifically, the glycoengineered P. pastoris system provides a balance between production efficiency and obtaining human-like post-translational modifications, which may be critical for proper folding and function .

• What purification methods are effective for recombinant HHIPL2?

  Purification of recombinant HHIPL2 typically involves:

  • Affinity chromatography: Using His-Tag or FC-Tag for selective capture . One-step homogeneous purification via affinity chromatography has been established for similar recombinant proteins .

  • Quality assessment: SDS-PAGE and Western blotting to confirm purity (typical commercial preparations specify >90% purity) .

  • Functional verification: Circular dichroism spectroscopy to confirm retention of secondary structure post-purification .

  A methodological approach would involve:

  1. Initial capture using affinity chromatography based on the fusion tag

  2. Potential intermediate purification using ion exchange chromatography

  3. Polishing step with size exclusion chromatography if needed

  4. Confirmation of purity by SDS-PAGE and activity by functional assays

• How can researchers verify the identity and integrity of recombinant HHIPL2?

  Comprehensive verification involves multiple complementary techniques:

  Verification Method	Application to HHIPL2	Expected Outcome
  SDS-PAGE	Molecular weight confirmation	Observed MW: ~81 kDa
  Western Blotting	Immunological confirmation	Specific band detection using anti-HHIPL2 antibodies
  Mass Spectrometry	Sequence verification	Peptide mapping matching theoretical sequence
  Circular Dichroism	Secondary structure analysis	Confirmation of properly folded protein
  Endotoxin Testing	Safety assessment	Endotoxin level within acceptable limits for experimental use

  Researchers should employ multiple methods to ensure both the identity and structural integrity of the recombinant protein, as proper folding is crucial for functional studies.

• What is known about HHIPL2 tissue expression and cellular localization?

  HHIPL2 is predicted to be located in the extracellular region . Tissue expression data from the Human Protein Atlas (available but not detailed in the search results) would provide comprehensive information about expression patterns across different organs and tissues . This extracellular localization is consistent with potential roles in intercellular signaling or matrix interactions. For experimental localization studies, researchers can employ:

  • Immunohistochemistry (IHC) using validated antibodies

  • Fluorescent protein fusion constructs for live-cell imaging

  • Subcellular fractionation followed by Western blotting

  • Secretion assays to confirm extracellular presence

• What methods can be used to assess recombinant HHIPL2 functionality?

  Since HHIPL2 is predicted to possess catalytic activity , functional assessment should focus on:

  • Enzyme activity assays: Based on predicted catalytic properties

  • Cell-based functional assays: Testing effects on appropriate cellular models

  • Binding assays: Identifying potential interaction partners

  • Structural analysis: Assessing proper folding via circular dichroism

  When designing experiments, researchers should consider that recombinant HHIPL2 has shown angiogenic potential in ex vivo chicken embryo models , suggesting it may influence vascular development pathways.

• What recombinant fragments or domains of HHIPL2 are available for research?

  Several recombinant fragments of human HHIPL2 have been produced for research:

  Fragment	Amino Acid Range	Applications	Source
  Control Fragment	aa 132-204	Blocking experiments, antibody validation	ThermoFisher
  Recombinant Fragment	aa 226-422	Immunogen for antibody production	Abbexa Ltd
  Full-length protein	Complete sequence	Functional studies	Creative Biolabs

  Researchers can select appropriate fragments based on their experimental needs, with options ranging from specific domains for antibody validation to the complete protein for functional studies.

Advanced Research Questions

• What are optimal conditions for expressing recombinant HHIPL2 in glycoengineered Pichia pastoris?

  Optimizing expression in glycoengineered P. pastoris requires systematic consideration of multiple parameters:

  • Vector design: Codon optimization for P. pastoris is critical, as demonstrated in similar recombinant protein production systems .

  • Promoter selection: The AOX1 (alcohol oxidase) promoter is commonly used for methanol-inducible expression.

  • Signal sequence: The α-mating factor prepro-sequence is frequently employed for efficient secretion.

  • Cultivation conditions:

    • Temperature: Typically 25-30°C, with potential for lower temperatures during induction

    • pH: Maintain between 5.0-6.0 using buffered media

    • Dissolved oxygen: Keep above 20% saturation

    • Carbon source: Initial glycerol batch followed by methanol induction

  • Process monitoring: Track growth via OD600, protein expression via SDS-PAGE, and glycosylation patterns

  A high-throughput process development (HTPD) approach, as described in bioprocess optimization literature, allows for efficient testing of multiple conditions in parallel .

• How can researchers modify HHIPL2 expression constructs using recombineering techniques?

  Recombineering (recombination-based genetic engineering) offers precise DNA manipulation capabilities for HHIPL2 construct modification:

  1. Design strategy:

     • Determine modification goals (tag addition, sequence alteration, domain deletion)

     • Choose appropriate recombineering system (λ Red system is commonly used)

  2. Primer design for targeting construct generation:

     • Design primers with 50 bp homology arms flanking the modification site

     • Include appropriate tags or modifications in the primer sequence

  3. Recombination procedure:

     • Express λ Red recombination proteins in E. coli

     • Transform with linear DNA containing desired modifications

     • Select recombinants using appropriate markers

  4. Verification:

     • PCR screening

     • Restriction analysis

     • Sequencing to confirm correct modifications

  For seamless modifications without selection markers, researchers can employ the two-step "hit and fix" method described in recombineering protocols .

• What approaches can be used to characterize the glycosylation profile of recombinant HHIPL2?

  As a glycoprotein produced in glycoengineered Pichia pastoris, HHIPL2's glycosylation pattern requires careful characterization:

  • Glycosite identification:

    • Bioinformatic prediction of N-linked (N-X-S/T) and O-linked glycosylation sites

    • Site-directed mutagenesis of predicted sites to confirm importance

  • Glycan profiling techniques:

    • MALDI-TOF mass spectrometry for glycan composition analysis

    • HILIC-UPLC for glycoform separation and quantification

    • Lectin microarrays for glycan structure screening

  • Comparative analysis:

    • Compare glycosylation between native and recombinant protein

    • Assess glycoform heterogeneity across production batches

  Recent approaches for controlling protein glycosylation include glycoengineering of expression hosts and upstream process optimization . These approaches are crucial for ensuring consistent glycoform production, which can significantly impact the protein's stability, half-life, and biological activity.

• How can researchers design domain-specific studies to elucidate HHIPL2 function?

  A systematic approach to understand HHIPL2 domain functions would include:

  1. Bioinformatic analysis:

     • Identify conserved domains through sequence comparison with better-characterized proteins

     • Predict functional regions based on structural modeling

  2. Domain-specific construct generation:

     • Create truncated constructs expressing individual domains

     • Generate point mutants targeting predicted catalytic residues

     • Design domain-swapping experiments with related proteins

  3. Functional characterization:

     • Express domain constructs using the recombinant protein production system

     • Perform binding assays to identify domain-specific interaction partners

     • Assess catalytic activity of wild-type vs. mutant constructs

  4. Cellular studies:

     • Examine domain-specific effects in cellular models

     • Use domain-blocking antibodies to inhibit specific functions

     • Perform complementation assays with domain mutants

  This systematic approach allows for precise mapping of structure-function relationships within the HHIPL2 protein.

• What are the challenges in scaling up recombinant HHIPL2 production for structural studies?

  Scaling up production for structural studies presents specific challenges:

  Challenge	Methodological Solutions	Implementation Strategies
  Consistent glycosylation	- Employ glycoengineered expression systems - Optimize culture conditions - Consider enzymatic deglycosylation	- Monitor glycoform distribution across batches - Implement statistical process control - Standardize critical process parameters
  Protein aggregation	- Screen buffer conditions - Add stabilizing agents - Optimize purification protocol	- Use high-throughput stability screening - Implement quality-by-design principles - Monitor aggregation with dynamic light scattering
  Yield optimization	- Bioreactor process development - Fed-batch strategies - Optimized induction protocols	- Develop scale-down models - Use design of experiments (DoE) approach - Implement process analytical technology
  Structural homogeneity	- Size exclusion chromatography - Ion exchange chromatography - Crystallization screening	- Pre-crystallization testing - Thermal shift assays - Circular dichroism monitoring

  Integrated continuous bioprocessing approaches offer advantages for scaling up, allowing smaller facilities and equipment footprints while facilitating rapid process development and scale-up .

• How can researchers investigate HHIPL2 interactions with potential binding partners?

  A comprehensive interaction study would involve:

  1. Candidate approach:

     • Based on sequence homology with HHIP, test interaction with Hedgehog pathway components

     • Co-immunoprecipitation with tagged recombinant HHIPL2

     • Proximity ligation assays in relevant cell types

  2. Unbiased screening:

     • Yeast two-hybrid screening with HHIPL2 as bait

     • Affinity purification-mass spectrometry (AP-MS)

     • Protein microarray screening

  3. Validation techniques:

     • Surface plasmon resonance (SPR) for quantitative binding parameters

     • Fluorescence resonance energy transfer (FRET) for interaction in living cells

     • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  4. Functional relevance:

     • Competition assays with predicted interacting proteins

     • Mutagenesis of predicted interaction interfaces

     • Cellular assays to assess functional consequences of disrupted interactions

  This multi-layered approach ensures both discovery and validation of physiologically relevant interaction partners.

• What methods can be used to assess the effect of point mutations on HHIPL2 activity?

  Evaluating the impact of point mutations requires a systematic workflow:

  1. Mutation selection:

     • Target conserved residues across species

     • Focus on predicted catalytic or binding sites

     • Design alanine scanning of functional domains

  2. Construct generation:

     • Use site-directed mutagenesis techniques

     • Apply recombineering methods for complex modifications

     • Generate multiple variants in parallel

  3. Expression and purification:

     • Express wild-type and mutant proteins under identical conditions

     • Purify using standardized protocols

     • Verify structural integrity via circular dichroism

  4. Functional comparison:

     • Enzymatic activity assays

     • Binding affinity measurements

     • Stability assessments via thermal shift assays

     • Cellular function testing in appropriate models

  5. Structural analysis:

     • Crystallography or cryo-EM if feasible

     • Molecular dynamics simulations

  This approach provides both functional and mechanistic insights into the roles of specific residues.

• How can HHIPL2 be incorporated into disease-related research models?

  To investigate potential disease associations of HHIPL2:

  1. Expression analysis in disease tissues:

     • Compare HHIPL2 expression in normal versus pathological samples

     • Analyze public datasets for expression correlations with disease progression

  2. Functional studies in disease models:

     • Overexpression/knockdown in relevant cell types

     • Addition of purified recombinant HHIPL2 to cell cultures

     • Ex vivo tissue models (leveraging known angiogenic potential)

  3. Animal model applications:

     • Genetic models (knockout/knockin)

     • Administration of recombinant protein

     • Therapeutic targeting using antibodies or small molecules

  4. Clinical correlation:

     • Analysis of genetic variations in patient populations

     • Development of biomarker applications

     • Exploration of therapeutic potential

  Given HHIPL2's predicted catalytic activity and extracellular localization, it may represent an accessible target for both diagnostic and therapeutic development in relevant disease contexts.