RLBP1 Human, sf9

What is RLBP1 protein and what are its functional characteristics?

RLBP1, also known as Cellular retinaldehyde-binding protein (CRALBP), is a 36-kD water-soluble protein that functions as a critical component of the visual cycle. It specifically transports 11-cis-retinal or 11-cis-retinaldehyde as physiologic ligands, making it essential for proper visual function . The recombinant human RLBP1 protein produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 326 amino acids (1-317) with a molecular mass of 37.5kDa, though it typically appears at approximately 28-40kDa on SDS-PAGE analysis due to glycosylation patterns . When expressed recombinantly, RLBP1 is commonly fused to a 6 amino acid His-Tag at the C-terminus to facilitate purification via chromatographic techniques .

The amino acid sequence of recombinant RLBP1 is well-characterized and includes specific domains involved in retinoid binding and protein-protein interactions essential for visual cycle functionality. These structural features are critical for researchers to understand when designing experiments involving RLBP1 manipulation or assessment .

How should RLBP1 Human, sf9 be handled in laboratory settings?

For optimal experimental results, RLBP1 protein solutions (typically at 0.5mg/ml concentration) should be stored in Phosphate Buffered Saline (pH 7.4) containing 20% glycerol . Short-term storage (2-4 weeks) can be maintained at 4°C if the entire vial will be used within that timeframe. For longer periods, the protein should be stored frozen at -20°C, while long-term storage is best achieved at -70°C, where recombinant proteins remain stable for up to 1 year from the receipt date .

To maximize protein stability during extended storage periods, researchers should consider adding a carrier protein (0.1% HSA or BSA) . It is crucial to avoid repeated freeze-thaw cycles as these can significantly compromise protein integrity and experimental reproducibility. When designing experiments, schedule work to minimize the number of freeze-thaw cycles the protein undergoes .

What disease models are associated with RLBP1 mutations?

RLBP1 variants are associated with a spectrum of inherited retinal dystrophies (IRDs), each with distinct clinical presentations and progression patterns . The major RLBP1-associated disorders include:

• Retinitis Punctata Albescens (RPA): Characterized by night blindness beginning in childhood and typically progressing to legal blindness by approximately 40 years of age. This represents a more moderate progression timeline compared to other RLBP1-associated disorders .

• Bothnia Dystrophy (BD): Distinguished by early involvement of the macula, which leads to central vision loss in addition to the night blindness symptoms common to all RLBP1-associated conditions .

• Newfoundland Rod-Cone Dystrophy (NFRCD): Presents with night blindness from infancy and severe visual loss beginning from approximately 20 years of age, representing a more aggressive progression than RPA .

Understanding these distinct phenotypes is essential for researchers designing targeted therapies or conducting natural history studies of RLBP1-associated conditions .

What approaches are used for RLBP1 gene replacement studies?

RLBP1 gene replacement studies typically employ adeno-associated viral (AAV) vectors to deliver functional copies of the gene to affected tissues. Current research has demonstrated success with AAV2/5 vectors expressing RLBP1 under control of a ubiquitous promoter (such as CAG) . The methodological workflow for such studies generally follows these steps:

• Vector design and production: Generation of an AAV2/5-CAG-RLBP1 vector that expresses functional CRALBP protein.

• Expression verification: Confirmation of RLBP1 and CRALBP expression through appropriate molecular and biochemical assays.

• Delivery method optimization: For retinal studies, subretinal injections are typically performed.

• Functional assessment: Visual cycle kinetics and outer retinal function are evaluated following photobleaching and dark-adaptation protocols.

• Comparative analysis: Results are compared between treated subjects and controls to quantify therapeutic efficacy .

This approach has shown promising results in Rlbp1-/- mice, where the expressed CRALBP protein improved visual cycle kinetics and outer retinal function, suggesting potential therapeutic applications for human patients with RLBP1 mutations .

How are iPSC-derived retinal models developed for RLBP1-associated disorders?

Patient-specific induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) models represent a cutting-edge approach to studying RLBP1-associated retinal dystrophies. The development process involves:

• Patient selection: Identifying individuals with characterized RLBP1 variants representing different clinical forms (RPA, BD, NFRCD).

• iPSC generation: Reprogramming patient-derived somatic cells into pluripotent stem cells.

• Directed differentiation: Guiding iPSCs toward retinal lineages, specifically RPE cells.

• Characterization:

  • Morphological assessment through immunofluorescence studies and electron microscopy

  • Functional evaluation via phagocytosis and secretion assays

  • Biochemical analysis of retinoid levels

• Validation: Comparison to control RPE derived from individuals without RLBP1 mutations .

These models enable researchers to study disease mechanisms in patient-specific cellular contexts and serve as platforms for proof-of-concept gene replacement studies. Recent research has demonstrated successful transduction of these models with therapeutic vectors and subsequent evaluation of CRALBP expression and retinoid levels post-treatment .

What outcome measures are most effective for evaluating RLBP1 therapy efficacy?

Comprehensive evaluation of RLBP1-targeted therapies requires both objective clinical measures and patient-reported outcomes (PROs). Recent research has validated the effectiveness of two non-disease-specific PRO instruments in RLBP1 retinal dystrophy patient populations:

• National Eye Institute Visual Function Questionnaire-25 (VFQ-25): This instrument evaluates multiple domains of visual function, with lowest scores in RLBP1 patients typically seen in distance activities (39.2–49.0) and peripheral vision (37.5–52.4) .

• Low Luminance Questionnaire (LLQ): This complementary instrument captures aspects not covered by VFQ-25, including responses to bright and dim lighting conditions and mobility challenges .

Rasch analysis of both instruments has shown that they effectively cover the distribution of person function in RLBP1 patients, indicating appropriate item difficulties for this population . Longitudinal studies have documented statistically significant declines in peripheral vision (measured by both VFQ-25 and LLQ), distance vision (VFQ-25), and adaptation to extreme lighting conditions (LLQ) over 2-2.5 years of follow-up .

These PRO instruments show strong correlation with each other (Pearson correlation coefficients ranging from 0.81 to 0.91 across different time points) and with clinical measures of visual function, making them valuable tools for comprehensive therapy assessment .

What are the critical factors in designing experiments with RLBP1 Human, sf9?

When designing experiments using recombinant RLBP1 from sf9 cells, researchers should consider several critical factors:

• Protein purity verification: Before experimental use, verify protein purity (>90% as determined by SDS-PAGE) to ensure experimental validity .

• Buffer compatibility: The standard formulation containing Phosphate Buffered Saline (pH 7.4) and 20% glycerol may interact with certain experimental reagents. Consider buffer exchange if necessary, but document and validate any modifications to the storage solution .

• Protein functionality assays: Prior to complex experiments, verify the retinoid-binding capacity of the recombinant protein using established biochemical assays.

• His-tag considerations: The C-terminal 6xHis-tag may affect protein interactions in some experimental contexts. Control experiments comparing tagged and untagged versions may be necessary for certain interaction studies .

• Glycosylation impact: The glycosylation pattern of sf9-expressed RLBP1 differs from mammalian expressions, which may affect protein behavior in certain assays. Consider this when interpreting results, especially in protein-protein interaction studies .

How can researchers effectively model RLBP1-associated disease progression?

Modeling RLBP1-associated disease progression requires multi-modal approaches integrating both clinical metrics and patient-reported outcomes. Effective research strategies include:

• Longitudinal assessment design: Studies should incorporate regular follow-up intervals (baseline, 1/1.5, 2/2.5, and 3/3.5 years) to capture meaningful changes over time .

• Domain-specific measurement: Focus particularly on peripheral vision, distance activities, and adaptation to extreme lighting conditions, as these domains show statistically significant declines over time in RLBP1 retinal dystrophy patients .

• Combined functional assessment: Integrate both objective clinical measures and validated PRO instruments (VFQ-25 and LLQ) to comprehensively assess disease impact .

• Phenotype stratification: Account for different clinical presentations (RPA, BD, NFRCD) in analysis, as progression rates and patterns may vary significantly between these phenotypes .

• Statistical approach: Use Rasch analysis for PRO data to properly calibrate item and person measures, enabling more sensitive detection of disease progression .

This multi-dimensional approach yields more comprehensive insights into disease progression than relying solely on clinical or structural measurements.

What are the challenges in analyzing RLBP1 isoform expression patterns?

Recent research has identified that RLBP1 gene replacement studies must account for the existence of two CRALBP isoforms that are differentially expressed in murine retina and human iPSC-derived retinal models . The analytical challenges this presents include:

• Isoform discrimination: Developing antibodies or molecular probes that can distinguish between closely related CRALBP isoforms.

• Expression pattern characterization: Establishing the normal temporal and spatial expression patterns of each isoform in different retinal cell types.

• Functional redundancy assessment: Determining whether the isoforms have overlapping or distinct functions in the visual cycle.

• Species differences: Accounting for potential differences in isoform expression and function between mouse models and human tissues.

• Therapeutic implications: Evaluating whether gene replacement strategies need to target specific isoforms or can succeed with expression of either/both forms .

Addressing these analytical challenges requires combined approaches using transcriptomic, proteomic, and functional assays in both animal models and human-derived tissues.

How can patient-reported outcomes be integrated with objective measures in RLBP1 research?

Integrating PROs with objective clinical measures represents a methodological challenge in RLBP1 research. Effective approaches include:

• Correlation analysis: Studies have demonstrated strong correlations between PRO composite scores and clinical measures of visual function. These correlations provide validation of PRO instruments while offering complementary information about disease impact .

• Domain-specific matching: Specific PRO domains can be matched with corresponding objective measures (e.g., peripheral vision scores from VFQ-25/LLQ with visual field measurements) to provide multi-dimensional assessment of particular visual functions .

• Statistical integration: Using multivariate statistical approaches to develop combined endpoints that incorporate both PRO data and objective measurements, potentially increasing sensitivity to detect therapeutic effects.

• Longitudinal modeling: Developing models that predict how changes in objective measures will translate to PRO outcomes over time, allowing for better clinical trial planning and interpretation .

The demonstrated correlation between VFQ-25 and LLQ (with correlation coefficients ranging from 0.81 to 0.91) supports the validity of these instruments, while their complementary nature ensures comprehensive assessment of visual function in RLBP1 patients .

What are the emerging techniques for studying RLBP1 function in visual cycle metabolism?

Emerging techniques for investigating RLBP1's role in visual cycle metabolism include:

• Cryo-electron microscopy: Providing high-resolution structural insights into RLBP1-retinoid interactions and conformational changes during the visual cycle.

• Live-cell imaging: Using fluorescently tagged RLBP1 variants to track protein dynamics within photoreceptors and RPE cells during light and dark adaptation.

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics to comprehensively map RLBP1's role in retinoid metabolism networks.

• CRISPR-based functional genomics: Creating precise mutations to map structure-function relationships in RLBP1 and identify critical residues for therapeutic targeting.

• Computational modeling: Developing in silico models of the visual cycle that incorporate RLBP1 dynamics to predict the impact of mutations and potential therapeutic interventions.

These advanced techniques promise to reveal new insights into RLBP1 biology and identify novel therapeutic approaches for RLBP1-associated retinal dystrophies.

What are the potential applications of RLBP1 Human, sf9 beyond retinal research?

While RLBP1 is primarily studied in the context of retinal function and disease, recombinant RLBP1 from sf9 expression systems has potential applications in broader research contexts:

• Drug discovery platform: As a target for high-throughput screening of compounds that might stabilize mutant RLBP1 proteins or enhance retinoid binding.

• Biomarker development: As a standard for developing quantitative assays of RLBP1 levels in biological fluids for potential diagnostic applications.

• Structural biology research: As a model system for studying protein-lipid interactions, given RLBP1's well-characterized binding to hydrophobic retinoid molecules.

• Protein engineering: As a scaffold for developing modified lipid-binding proteins with novel specificities or functions.

• Immunological studies: For developing and characterizing antibodies against different epitopes or conformational states of RLBP1, which may have diagnostic applications.

These diverse applications highlight the value of high-quality recombinant RLBP1 preparations beyond their immediate relevance to inherited retinal disease research.