Recombinant Macaca fascicularis Leucine-rich repeat-containing protein 53 (LRRC53)

What is the structural composition of Macaca fascicularis LRRC53?

Macaca fascicularis Leucine-rich Repeat-containing Protein 53 (LRRC53) is a 510 amino acid protein characterized by multiple leucine-rich repeat domains. The commercially available recombinant form contains the full-length protein (amino acids 1-510) with the complete amino acid sequence: MLQLVAACPESCVVCTKDVTLCHQLTYIVAAPMTTRVLIITDGYLSSIESTNLSLLFNLALLSLSRNGIEDVQEDALDGLTMLRTLLLEHNQISSSSLTDHTFSKLHSLQVLVLSNNALRTLRGSWFRNTRGLTRLQLDGNQITNLTDSSFGGTNLHSLRHLDLSNNFISYIGKDAFRPLPQLQEVDLSRNRLAHMPDVFTPLKQLIHLSLDKNQWSCTCDLHPLARFLRNYIKSSAHTLRNAKDLNCQPSTAAVAAAQSVLRLSETNCDPKAPNFTLVLKDRSPLLPGQDVALLTVLGFAGAVGLTCLGLVVFNWKLQQGKANEHTSENLCCRTFDEPLCAHGARNYHTKGYCNCHLTQENEIKVMSIVGSRKEMPLLQENSHQATSASESTTLDGSFRNLKKKDHGVGSTLFCQDGRLLHSRCSQSPGNTTAFNEAGLLTTYNSRKVQKLRNLESGEVLPQTLPHHIIRTEDISSDTFRRRYAIPTSALAGESLEKHLTNESCLHTLN .

The protein contains characteristic leucine-rich repeat domains that are typically involved in protein-protein interactions and may play roles in signal transduction or other cellular processes.

How is recombinant Macaca fascicularis LRRC53 typically produced for research applications?

Recombinant Macaca fascicularis LRRC53 is typically expressed in prokaryotic expression systems, particularly E. coli, for research applications . The protein is engineered to include an N-terminal His-tag to facilitate purification using affinity chromatography. After expression, the protein undergoes purification processes to achieve >90% purity as determined by SDS-PAGE analysis . The final product is typically provided as a lyophilized powder in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . This production method allows for consistent quality and quantity of the protein for experimental use.

What are the optimal storage and reconstitution conditions for recombinant LRRC53?

For optimal preservation of recombinant Macaca fascicularis LRRC53 activity, the protein should be stored at -20°C to -80°C upon receipt . Aliquoting is necessary for multiple uses to avoid repeated freeze-thaw cycles, which can degrade protein structure and function. For short-term use, working aliquots can be stored at 4°C for up to one week .

For reconstitution, it is recommended to:

• Briefly centrifuge the vial prior to opening

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (typically 50%) for long-term storage

• Prepare small aliquots to minimize freeze-thaw cycles

This methodological approach maximizes protein stability and experimental reproducibility.

How does sequence homology between Macaca fascicularis LRRC53 and human LRRC53 impact experimental design?

When using Macaca fascicularis LRRC53 as a model for human studies, researchers must consider the sequence homology between species. Available data shows that human LRRC53 has relatively low sequence identity with other mammalian orthologs (29% with mouse and rat) . While specific homology data between human and Macaca fascicularis LRRC53 is not directly provided in the search results, the general phylogenetic proximity of macaques to humans suggests potentially higher conservation than with rodents.

Experimental designs should account for these sequence differences by:

• Performing protein alignment analyses prior to designing antibodies or interaction studies

• Validating antibody cross-reactivity between species if using anti-human antibodies

• Considering potential functional differences when extrapolating results between species

• Using appropriate negative controls to verify specificity of observed interactions

Researchers should be aware that despite cynomolgus macaques being phylogenetically closer to humans than rodents, significant protein-specific differences may exist that can impact experimental outcomes and interpretation .

What experimental considerations are important when using recombinant LRRC53 in immunological research?

When designing immunological experiments with recombinant Macaca fascicularis LRRC53, several important considerations should be taken into account:

• Expression system impact: The E. coli-expressed recombinant protein lacks eukaryotic post-translational modifications, which may affect protein folding, activity, and immunogenicity compared to the native protein .

• Tag interference: The His-tag at the N-terminus may influence protein function or antibody recognition in certain experimental settings. Control experiments with tag-cleaved protein may be necessary for validation .

• Species-specific immune responses: When studying immune responses in different animal models, researchers should consider that Macaca fascicularis has a distinct MHC polymorphism that influences antigen presentation and immune responses . The MHC genotype of experimental animals should be characterized when assessing immune responses to LRRC53.

• Endotoxin contamination: Proteins expressed in E. coli may contain endotoxin contaminants that can trigger innate immune responses, potentially confounding results in immunological experiments. Endotoxin testing and removal may be necessary .

These methodological considerations help ensure experimental validity and reproducibility in immunological research involving recombinant LRRC53.

What methods are optimal for detecting protein-protein interactions involving LRRC53?

Given the leucine-rich repeat structure of LRRC53, which typically mediates protein-protein interactions, several methods can be optimized for studying its binding partners:

• Co-immunoprecipitation (Co-IP):

  • Use anti-His antibodies to pull down His-tagged LRRC53 and identify interacting proteins

  • Alternatively, use anti-LRRC53 specific antibodies when available

  • Include appropriate controls: tag-only proteins, irrelevant proteins of similar size

• Pull-down assays:

  • Immobilize purified recombinant LRRC53 on Ni-NTA or other affinity resins

  • Incubate with cell lysates or purified candidate proteins

  • Wash extensively to remove non-specific binding

  • Elute and analyze bound proteins by mass spectrometry or immunoblotting

• Surface Plasmon Resonance (SPR):

  • Immobilize LRRC53 on a sensor chip

  • Flow potential binding partners over the surface

  • Measure real-time binding kinetics

  • Calculate association and dissociation constants

• Yeast Two-Hybrid screening:

  • Use LRRC53 as bait to identify novel interacting partners

  • Validate findings with orthogonal methods listed above

These methodological approaches should be adapted based on the specific experimental questions and available resources.

How can researchers address challenges in functional characterization of LRRC53 given limited existing literature?

The functional characterization of LRRC53 presents challenges due to limited existing literature specifically focusing on this protein. A comprehensive approach to address these limitations includes:

• Comparative genomics and in silico analysis:

  • Perform phylogenetic analysis comparing LRRC53 across species

  • Use structural prediction tools to identify functional domains

  • Apply protein-protein interaction prediction algorithms to identify potential binding partners

  • Analyze tissue expression patterns using available transcriptomic datasets

• Generation of cellular models:

  • Develop LRRC53 knockout and overexpression systems in relevant cell lines

  • Use CRISPR-Cas9 to introduce tagged versions at endogenous loci

  • Create domain deletion mutants to identify critical functional regions

• Multi-omics approach:

  • Perform proteomics analysis of LRRC53 interactome under different conditions

  • Analyze transcriptome changes following LRRC53 modulation

  • Investigate post-translational modifications using mass spectrometry

• Collaborative cross-disciplinary research:

  • Establish collaborations with structural biologists for crystallography studies

  • Partner with immunologists to investigate potential immune functions

  • Engage computational biologists for systems-level analysis

This methodological framework provides a systematic approach to functional characterization despite the current knowledge gaps.

What are the considerations for developing a robust assay to measure LRRC53 activity?

Developing a robust assay to measure LRRC53 activity requires careful consideration of protein characteristics and potential functions. Since leucine-rich repeat proteins often function in signaling pathways or protein-protein interactions, assay development should consider:

• Activity definition considerations:

  • Determine whether to measure binding activity, signaling outcomes, or structural changes

  • Identify cellular contexts where LRRC53 is physiologically relevant

  • Consider whether activity requires co-factors or post-translational modifications absent in recombinant protein

• Assay methodology options:

  • Binding assays using fluorescence polarization or FRET if interacting partners are known

  • Cell-based reporter assays if LRRC53 impacts specific signaling pathways

  • Structural assays (circular dichroism, thermal shift) to measure conformational changes upon ligand binding

• Validation strategy:

  • Include positive and negative controls (related proteins with known functions)

  • Test activity across concentration ranges to establish dose-response relationships

  • Confirm specificity using competitive inhibitors or blocking antibodies

  • Assess reproducibility across different protein batches and experimental conditions

• Physiological relevance verification:

  • Compare results using recombinant protein versus endogenously expressed LRRC53

  • Validate findings in primary cells from Macaca fascicularis

  • Consider species differences when extrapolating findings to human systems

This comprehensive approach facilitates development of meaningful activity assays despite limited prior characterization of LRRC53 function.

How can researchers address the MHC polymorphism impact when studying immune responses to LRRC53 in Macaca fascicularis models?

When studying immune responses to LRRC53 in Macaca fascicularis models, researchers must address the significant impact of MHC polymorphism, which can dramatically influence experimental outcomes. A methodological approach includes:

• MHC genotyping of experimental animals:

  • Characterize MHC class I and II alleles in all experimental animals using sequencing techniques

  • Group animals based on MHC haplotypes for balanced experimental design

  • Consider using animals from defined populations with limited MHC diversity (e.g., Mauritian origin) for reduced variability

• Epitope prediction and validation:

  • Use computational tools to predict LRRC53 epitopes that bind to common Macaca fascicularis MHC molecules

  • Experimentally validate binding using in vitro MHC-peptide binding assays

  • Map T cell responses to specific epitopes using ELISpot or intracellular cytokine staining

• Experimental design considerations:

  • Match treatment and control groups for MHC haplotypes to minimize confounding effects

  • Include sufficient animal numbers to account for MHC diversity

  • Consider using MHC-matched animals for adoptive transfer experiments

• Data analysis approach:

  • Stratify immunological readouts based on MHC genotypes

  • Apply multivariate analysis to distinguish MHC effects from experimental variables

  • Report MHC types alongside experimental results to facilitate interpretation

This framework addresses the established fact that MHC polymorphism significantly influences immune responses in nonhuman primate models, as documented in infectious disease and vaccine studies using Macaca fascicularis .

What quality control parameters should be assessed before using recombinant LRRC53 in experiments?

Before using recombinant Macaca fascicularis LRRC53 in experiments, researchers should assess several critical quality control parameters to ensure experimental reproducibility and valid results:

• Purity assessment:

  • Verify >90% purity by SDS-PAGE analysis as specified in product information

  • Consider additional analytical methods like size exclusion chromatography for aggregation analysis

  • Perform mass spectrometry to confirm protein identity and integrity

• Functional verification:

  • Assess protein folding using circular dichroism or fluorescence spectroscopy

  • Verify tag accessibility via small-scale pull-down if His-tag functionality is required

  • Perform binding assays with known interactors if available

• Contamination testing:

  • Test for endotoxin contamination, especially critical for immunological experiments

  • Assess microbial contamination if protein will be used in long-term cell culture

  • Verify buffer composition matches specifications

• Batch consistency analysis:

  • Compare new batches with previously validated lots

  • Maintain reference samples from effective batches

  • Document lot-specific information including expression date and conditions

These methodological quality control steps should be documented and included in experimental methods sections to enhance reproducibility and scientific rigor.

What are the most effective approaches for optimizing antibody development against Macaca fascicularis LRRC53?

Developing effective antibodies against Macaca fascicularis LRRC53 requires strategic approaches that account for protein characteristics and experimental applications:

• Antigen design considerations:

  • Use full-length protein for polyclonal antibody generation

  • For monoclonal antibodies, select unique epitopes with low homology to other LRR proteins

  • Consider synthetic peptides from predicted surface-exposed regions

  • Evaluate cross-reactivity potential with human LRRC53 if dual-species recognition is desired

• Host selection strategy:

  • Choose host species phylogenetically distant from primates to maximize immunogenicity

  • Consider rabbits for polyclonal antibodies or mice for monoclonal development

  • Evaluate multiple host species if initial attempts yield poor responses

• Validation methodology:

  • Test antibody specificity using Western blot against recombinant protein

  • Validate using LRRC53-expressing cells versus LRRC53-knockout controls

  • Perform immunoprecipitation to confirm native protein recognition

  • Evaluate cross-reactivity with related LRR proteins

• Application-specific optimization:

  • For immunohistochemistry: test various fixation methods and antigen retrieval techniques

  • For flow cytometry: optimize antibody concentration and staining buffers

  • For neutralization studies: screen for antibodies targeting functional domains

This methodological framework enhances the likelihood of generating high-quality antibodies for LRRC53 research applications.

What methodological approaches can address the challenges of comparative studies between macaque and human LRRC53?

Comparative studies between macaque and human LRRC53 present unique challenges that require specific methodological approaches:

• Sequence and structural analysis framework:

  • Perform detailed sequence alignment to identify conserved and divergent regions

  • Use homology modeling to predict structural differences

  • Compare post-translational modification sites between species

  • Analyze conservation of binding interfaces for potential interaction partners

• Expression system standardization:

  • Express both proteins in identical systems (e.g., same E. coli strain or mammalian cell line)

  • Use identical tags and purification protocols

  • Characterize both proteins using the same analytical methods

  • Process and store proteins under identical conditions

• Functional comparison methodology:

  • Develop parallel assays with identical conditions for both proteins

  • Use chimeric constructs to identify functionally divergent domains

  • Test interaction with conserved binding partners from both species

  • Measure binding kinetics using surface plasmon resonance or similar techniques

• Translational research considerations:

  • Evaluate both proteins in identical cellular contexts

  • Consider the impact of species-specific co-factors on functional outcomes

  • Document species differences alongside similarities

  • Explicitly state limitations when extrapolating between species

These methodological approaches help researchers navigate the challenges inherent in cross-species protein comparisons while maximizing translational value between macaque models and human applications .

Key Properties of Recombinant Macaca fascicularis LRRC53 Protein

Comparison of Human LRRC53 Ortholog Sequence Identity