Recombinant Human Bestrophin-1 (BEST1)
Description
Overview of Recombinant Human Bestrophin-1
Recombinant Human BEST1 is produced through heterologous expression systems to study its channel activity, structure, and disease-associated mutations. Key features include:
Expression Systems
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E. coli: Used by ProSpec Bio and Creative BioMart to produce truncated BEST1 (residues 292–585) with a 36 kDa molecular weight .
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Wheat germ: Employed by Abcam for full-length BEST1 (1–604 aa), retaining native-like post-translational modifications .
Purification and Stability
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Storage: Stable at -20°C in Tris-HCl buffer (pH 8.0) with 10% glycerol; carrier proteins (e.g., BSA) recommended for long-term storage .
Channel Activity
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Acts as a calcium- and volume-regulated chloride/bicarbonate channel .
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Pathological relevance: Loss of CaCC activity in RPE correlates with Best disease phenotypes .
Disease Modeling
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Y227N mutation: Reduces protein half-life in testis, causing reproductive deficits in knock-in mice .
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P274R mutation: Abolishes CaCC currents in patient-derived RPE cells, reversible via viral gene supplementation .
Gene Therapy
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Loss-of-function mutations: Rescue achievable with wild-type BEST1 overexpression (e.g., AAV-mediated delivery) .
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Gain-of-function mutations: Require CRISPR/Cas9 knockdown of mutant alleles alongside gene augmentation .
Drug Development
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Recombinant BEST1 enables high-throughput screening for channel modulators .
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Challenges include mutation-specific rescue strategies and avoiding dominant-negative effects in heteromeric channels .
Future Directions
Product Specs
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Target Background
- hBest1's channel activity in human retinal pigment epithelium is significantly enhanced by adenosine triphosphate in a dose-dependent manner PMID: 30087350
- This study identified two recurrent genetic variations of BEST1 in two Chinese patients with either juvenile-onset BVMD or adult-onset BVMD. These findings expand the mutation spectrum of BEST1 and can be helpful for genetic counseling and prenatal diagnoses of patients with BVMD. PMID: 29115605
- These results highlight the critical role of BESTROPHIN1 in mediating the long-hypothesized Ca(2+)-dependent Cl(-) current in retinal pigment epithelium, which is the central disease-causing mechanism of BEST1 mutations. PMID: 29063836
- We identified a highly recognizable and reproducible retinal phenotype associated with a specific BEST1 mutation-p.Ala243Val in vitelliform macular dystrophy. PMID: 28225368
- We identified a consanguineous family with five affected individuals diagnosed with autosomal recessive bestrophinopathy and four confirmed carriers. Their pedigree indicated dominant inheritance with incomplete penetrance. PMID: 28481155
- Our research demonstrated that the two novel combinations of compound heterozygous mutations p.R141H/p.M325T and p.R141H/p.I201T in the BEST1 gene can also result in the autosomal recessive bestrophinopathy phenotype. PMID: 26333019
- The findings in this family emphasize the previously observed variability in clinical manifestations associated with BEST1-linked autosomal dominant vitreoretinochoroidopathy (ADVIRC) and the significance of FAF and NIA imaging. Cystoid macular edema and vascular leakage can be effectively managed using dorzolamide. PMID: 26771239
- Genetic analysis revealed single heterozygous BEST1 mutations in 13 patients and compound heterozygous mutations in 3 patients with Best vitelliform macular dystrophy. In patients with autosomal recessive bestrophinopathy, biallelic mutations were found in 13 probands and single mutant alleles in six patients. In total, 36 disease-causing variants (20 novel mutations) of the BEST1 gene were identified. PMID: 28687848
- Mutations in BEST1 exhibit variable penetrance and expressivity and can be uniocular. PMID: 27287821
- We identified 7 BEST1 variants, including 2 novel ones, in 9 Japanese patients diagnosed with autosomal recessive bestrophinopathy. PMID: 27163236
- Of the 225 genetic tests conducted, 150 were for recessive IRD, and 75 were for dominant IRD. A positive molecular diagnosis was established in 70 (59%) probands with recessive IRD and 19 (26%) probands with dominant IRD. We identified 32 novel variants; among them, 17 sequence changes in four genes (ABCA4, BEST1, PRPH2, and TIMP3) were predicted to be possibly or probably damaging. PMID: 28005406
- We describe the atypical phenotype and high intrafamilial variability associated with a new mutation in the BEST1 gene within an Italian family affected with Best vitelliform macular dystrophy. PMID: 26807628
- A clinical picture resembling autosomal recessive bestrophinopathy can also be caused by a single heterozygous mutation in the BEST1 gene, such as the c.614T>C (p.I205T) variant observed in this family. PMID: 26716959
- The secondary structure of Best1 and the influence of calcium have been characterized. PMID: 27768912
- BVMD can co-occur with other ocular disorders like ACG and FCE. We also identified 2 novel disease-causing mutations (p.Thr307Asp, p.Arg47His) in the BEST1 gene, one of which (p.Arg47His) has been reported in adult-onset vitelliform macular dystrophy (AVMD). PMID: 27078032
- Two previously unreported disease-associated variants in the BEST1 gene (p.Gly15Arg and p.Arg105Gly) were identified in Slovenian patients with Best disease. PMID: 27775230
- Nuclear spheres modulate the expression of BEST1 and GADD45G. PMID: 26521045
- Progressive posterior chorioretinal changes occurred over time in the initial ADVIRC proband, leading to visual loss. The causative mutation in this patient resides within the transmembrane domain of the BEST1 protein, with unclear functional consequences. PMID: 26849243
- HEK293T cells transfected with the identified BEST1 mutant exhibited significantly smaller currents compared to those transfected with the wild-type gene, while cells cotransfected with mutant and wild-type BEST1 showed good chloride conductance. PMID: 26720466
- Bestrophin 1 plays an essential role in volume regulation within human retinal pigment epithelium cells. PMID: 25941382
- We report a novel mutation within the BEST1 gene in the heterozygous form, leading to vitelliform lesions and secondary neovascularization that was successfully treated in a child with a course of bevacizumab. PMID: 25265375
- Dysfunction of bestrophin 1 can disrupt normal ion and fluid transport by the retinal pigment epithelium (RPE), interfering with, or even disrupting, direct interactions between the RPE and photoreceptors. PMID: 24328569
- We identified two novel BEST1 mutations and associated clinical observations in two unrelated patients with Best vitelliform macular dystrophy. PMID: 25936525
- Twelve distinct variants, including two novel ones (p.S7N and p.P346H), were identified in 13 Japanese families diagnosed with Best's vitelliform macular dystrophy. PMID: 26201355
- Genetic sequence analysis of BEST1 is crucial for diagnosing Best vitelliform dystrophy, especially in atypical cases, and contributes to our understanding of this disease. PMID: 26099059
- Best1 I366fsX18 differs from Best1 in that it lacks most of the cytosolic C-terminal domain, suggesting that the loss of this region contributes significantly to the pathogenesis of ARB in this patient. PMID: 26200502
- We describe three novel BEST1 mutations, highlighting that numerous deleterious variants in BEST1 leading to haploinsufficiency remain unidentified. PMID: 25545482
- BEST1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium. PMID: 25878489
- The characteristics and combinations of different BEST1 mutations, as well as epistatic effects, can influence phenotype expression in Chinese patients with bestrophinopathy. PMID: 25489231
- We describe the clinical and genetic features of a young male diagnosed with autosomal recessive bestrophinopathy associated with angle-closure glaucoma resulting from a novel homozygous mutation in BEST1. PMID: 24859690
- Bestrophin-1 functions as an intracellular Cl channel that facilitates the accumulation and release of Ca(2+) from stores by conducting the counterion for Ca(2+). PMID: 24664688
- Our current and previous findings indicate that mislocalization of Best1 is not a universal feature of any individual bestrophinopathy. PMID: 24560797
- Results show that different mutations in Best1 exert differential effects on its localization, and this effect varies depending on the presence or absence of wild-type (WT) Best1. PMID: 23825107
- Best1V1 and Best1V1Deltaex2 formed Ca2+-activated Cl-channels, indicating that the N-terminus is not essential for channel function. Cells expressing Best1V2 lacked detectable Ca2+-activated Cl-currents, highlighting a crucial role for splicing of the C-terminus. PMID: 24341532
- Given relatively well-preserved retinal function, autosomal recessive bestrophinopathy may be a suitable initial candidate among BEST1-related ocular conditions for gene replacement therapy. PMID: 24345323
- Three basolateral sorting motifs may be involved in proper Best1 basolateral localization. PMID: 23880862
- In a large consanguineous family, the cosegregation of p.C251Y with a consistent ocular phenotype in all 5 patients strongly suggests traits associated with BEST1 homozygous mutations: ARB, angle-closure glaucoma, hyperopia, and cataracts. PMID: 23823511
- Case Reports: siblings with a missense mutation in BEST1 presenting with retinoschisis and best vitelliform macular dystrophy. PMID: 23572118
- Autosomal recessive bestrophinopathy is a distinct phenotype caused by autosomal recessively inherited mutations in the BEST1 gene. PMID: 23290749
- Ten variants in the BEST1 gene were detected in a group of Italian patients with clinically apparent vitelliform macular dystrophy. PMID: 23213274
- This case of unilateral vitelliform phenotype further supports the notion that autosomal recessive bestrophinopathy represents a disease spectrum in terms of severity, age at onset, and heritability. PMID: 22584882
- Our findings expand the mutation spectrum of BEST1 in patients with Best disease. Our frequency estimate confirms that Best disease is one of the most prevalent causes of early macular degeneration. PMID: 22633354
- Two siblings carrying a homozygous Arg141His mutation developed symptoms of typical Best vitelliform dystrophy, while their parents exhibited clinical features of mild maculopathy. PMID: 21809908
- Autosomal recessive Best vitelliform macular dystrophy can be caused by the compound heterozygous mutation L41P and I201T in the BEST1 gene. PMID: 22422030
- Molecular screening of the candidate genes BEST1 and PRPH2 did not reveal any mutations. PMID: 22174098
- We report, for the first time, a phenotype-genotype correlation in a Czech patient with Best disease. PMID: 22448417
- Biallelic BEST1 sequence variants can be associated with at least two distinct phenotypes: Best vitelliform macular dystrophy and autosomal recessive bestrophinopathy. PMID: 22162627
- BEST1 regulated cell function in the cytosol by modulating calcium signaling. PMID: 22183384
- Bestrophinopathies constitute a group of inherited retinal disorders primarily caused by point mutations scattered throughout the BEST1 gene. PMID: 22183385
- In truncating BEST1 mutations, the null phenotype associated with ARB is attributed to a substantial decrease in BEST1 expression mediated by the nonsense-mediated decay (NMD) surveillance mechanism. PMID: 22199244
Q&A
What is Recombinant Human Bestrophin-1 (BEST1) and what is its physiological role?
Recombinant Human Bestrophin-1 (BEST1) is a protein encoded by the BEST1 gene that functions primarily as a calcium-activated chloride channel (CaCC) in the retinal pigment epithelium (RPE). The BEST1 protein is predominantly expressed in RPE cells, where it plays an indispensable role in mediating Ca²⁺-dependent Cl⁻ currents . When BEST1 is stimulated by calcium ions, it opens to allow chloride ions to flow into and out of the cell, which is critical for maintaining proper ion homeostasis in RPE cells .
This ion transport function is essential for supporting photoreceptor health and maintaining the visual cycle. Physiologically, BEST1 contributes to the light peak (LP) response in electrooculography (EOG), which is a diagnostic measure of RPE function. Patients with BEST1 mutations typically exhibit reduced LP, which is considered a pathognomonic phenotype in bestrophinopathies .
How are BEST1 mutations classified and what diseases are associated with them?
BEST1 mutations are classified based on their inheritance pattern and clinical presentation. Over 200 distinct mutations in the BEST1 gene have been identified and associated with at least five distinct retinal degeneration disorders:
| Disease Type | Inheritance Pattern | Clinical Features | Common Mutation Types |
|---|---|---|---|
| Best vitelliform macular dystrophy (BVMD) | Autosomal dominant | Central yellow "egg yolk" lesion in macula | Missense mutations |
| Autosomal recessive bestrophinopathy (ARB) | Autosomal recessive | Multifocal subretinal deposits, RPE changes | Null mutations (e.g., P274R) |
| Adult-onset vitelliform dystrophy (AVMD) | Autosomal dominant | Later onset, smaller vitelliform lesions | Missense mutations |
| Autosomal dominant vitreoretinochoroidopathy (ADVIRC) | Autosomal dominant | Peripheral retinal abnormalities | Splice-site mutations |
| Retinitis pigmentosa (RP) | Typically autosomal recessive | Progressive photoreceptor degeneration | Various types |
These mutations can affect protein folding, localization, or function. For example, the P274R mutation completely abolishes Ca²⁺-dependent Cl⁻ current in RPE cells, while the I201T mutation causes only partial impairment of channel function, correlating with the severity of the clinical phenotype in patients .
What experimental models are currently used to study BEST1 function?
Several experimental models have been developed to study BEST1 function:
| Model System | Applications | Advantages | Limitations |
|---|---|---|---|
| Patient-derived iPSC-RPE | Disease modeling, functional studies | Patient-specific mutations, physiologically relevant | Labor-intensive, variability between lines |
| HEK293 cell expression | Electrophysiology, protein trafficking | Ease of manipulation, high expression | Non-native cellular environment |
| Bacterial bestrophin homologs | Structural studies, basic channel properties | Easier to purify and crystallize | Evolutionary divergence from human BEST1 |
| Lipid bilayer systems | Isolated channel activity | Direct measurement of channel function | Lacks cellular regulatory mechanisms |
| Animal models (mouse, rat) | In vivo disease modeling | Systemic effects, retinal physiology | Species differences in retinal structure |
Patient-specific iPSC-based disease models have emerged as particularly valuable tools, as they allow researchers to directly correlate patient clinical phenotypes with electrophysiological properties in their RPEs and the structure-function relationships of BEST1 mutant channels .
How can we effectively measure and characterize Ca²⁺-dependent Cl⁻ currents mediated by BEST1?
Measurement of Ca²⁺-dependent Cl⁻ currents requires specialized electrophysiological techniques. The most direct and reliable method is whole-cell patch clamp recording, which can be performed on patient-derived iPSC-RPE cells or heterologous expression systems.
Methodological approach:
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iPSC-RPE preparation: Generate iPSC-RPEs from patient or control fibroblasts using established reprogramming protocols
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Patch clamp configuration: Utilize whole-cell patch clamp with carefully controlled intracellular Ca²⁺ concentrations
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Data acquisition parameters:
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Hold membrane potential at -50 to -80 mV
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Step voltage protocol ranging from -100 to +100 mV
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Internal solution containing varying free Ca²⁺ concentrations (0-1.2 μM)
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External solution with physiological or modified Cl⁻ concentrations
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The characteristic BEST1-mediated current shows outward rectification at positive potentials and is activated by intracellular Ca²⁺ in a concentration-dependent manner. Currents should be analyzed for peak amplitude, current-voltage relationships, and Ca²⁺ dose-response curves .
Alternative approaches include fluorescence-based chloride imaging with indicators such as MQAE or genetically encoded chloride sensors, which can provide spatial information about chloride flux across the RPE cell membrane.
What structural insights have been gained about BEST1 and how do they inform our understanding of mutation effects?
While the structure of human BEST1 has not been directly solved, significant structural insights have been obtained through bacterial homolog structures (KpBest and cBest1) and human homology models.
Key structural features of BEST1:
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Pentameric assembly: BEST1 functions as a homopentamer with five identical subunits arranged around a central pore
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Transmembrane domains: Each subunit contains multiple transmembrane helices forming the ion conduction pathway
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Calcium binding sites: Specific residues coordinate Ca²⁺ binding to activate the channel
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Neck region: Forms the narrowest part of the pore and is crucial for ion selectivity
Structural impact of mutations:
Different mutations can affect the structure and function of BEST1 in distinct ways:
Crystallographic studies with bacterial bestrophin homologs carrying equivalent mutations have provided direct evidence for these structural effects, complementing functional studies in patient-derived cells.
What approaches can be used to develop gene therapy strategies for BEST1-associated diseases?
Gene therapy represents a promising approach for treating BEST1-associated diseases, particularly for recessive forms like ARB. Research has demonstrated that viral expression of wild-type BEST1 can rescue the loss of Ca²⁺-dependent Cl⁻ current in patient-derived RPE cells carrying null mutations such as P274R .
Methodological considerations for BEST1 gene therapy:
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Vector selection: AAV vectors (particularly serotypes with RPE tropism like AAV2/4, AAV2/5, or AAV2/8) are preferred due to their safety profile and efficient transduction of RPE cells
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Construct design:
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Promoter: RPE-specific promoters (e.g., RPE65, BEST1, or VMD2) ensure targeted expression
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Coding sequence: Wild-type human BEST1 cDNA with codon optimization
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Regulatory elements: Enhancers and polyadenylation signals for stable expression
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Delivery route:
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Subretinal injection targets RPE cells directly
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Intravitreal delivery with enhanced viral vectors for better penetration
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Efficacy assessment:
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Functional rescue: Patch-clamp confirmation of restored Ca²⁺-dependent Cl⁻ currents
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Morphological improvement: Reduction in vitelliform lesions or subretinal deposits
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Visual function: Electroretinography (ERG), electrooculography (EOG), and visual acuity tests
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For dominant mutations, alternative approaches such as allele-specific knockdown using RNA interference or CRISPR-based gene editing may be necessary to selectively suppress the mutant allele while preserving wild-type function .
How can iPSC-RPE disease models be optimized for BEST1 research?
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Reprogramming and differentiation protocols:
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Integration-free reprogramming methods (Sendai virus, episomal vectors)
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Directed differentiation with defined factors (nicotinamide, Activin A, etc.)
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Purification based on RPE-specific markers (MITF, RPE65, BEST1)
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Validation of RPE identity and maturity:
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Disease phenotype characterization:
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Electrophysiological assessment of Ca²⁺-dependent Cl⁻ currents
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Ultrastructural analysis of RPE morphology
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Fluid and ion transport assays
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Response to pharmacological modulators
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Experimental controls:
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Isogenic controls generated by CRISPR correction of mutations
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Multiple independent iPSC lines from the same patient
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RPE cells from age-matched healthy donors
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The "disease-in-a-dish" approach with iPSC-RPEs enables prediction of the pathological potential of various BEST1 mutations and provides a platform for testing therapeutic interventions before clinical application .
What are the challenges in distinguishing phenotypes of dominant versus recessive BEST1 mutations?
Analyzing the functional and structural consequences of dominant versus recessive BEST1 mutations presents unique challenges:
| Challenge | Dominant Mutations | Recessive Mutations | Methodological Approach |
|---|---|---|---|
| Protein composition | Heterogeneous pentamers with mixed WT/mutant subunits | Homogeneous pentamers with all mutant subunits | Controlled expression systems with tagged WT/mutant proteins |
| Stoichiometry effects | Variable effects based on number of mutant subunits | All-or-none effects | Single-molecule imaging or biochemical quantification of subunit ratio |
| Rescue strategies | May require mutant suppression plus WT supplementation | Only require WT supplementation | Allele-specific targeting versus conventional gene supplementation |
| Structural analysis | Difficult to interpret due to heteropentamers | Cleaner interpretation with homopentamers | In vitro reconstitution with defined subunit composition |
As noted in the literature, recessive BEST1 mutations from ARB patients provide a "cleaner" system for analysis since only the mutant BEST1 proteins are present. In contrast, dominant mutations present the complexities of co-existing wild-type and mutant proteins, with pentameric channels potentially containing various combinations of wild-type and mutant protomers .
This heterogeneity makes it difficult to determine the precise ratio of wild-type to mutant proteins in patient cells and complicates interpretation of structural studies, which typically examine homopentamers rather than the heteropentamers likely present in patients with dominant mutations.
