Recombinant Human MANBA/Beta-Mannosidase His-tag Protein, CF
Recombinant Human MANBA/Beta-Mannosidase His-tag Protein, CF Summary
Product Specifications
The specific activity is >100 pmol/min/μg, as measured under the described conditions.
Ala19-Tyr879 & Glu20 - Ser878, with C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
10520-GH
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 25 mM MES, pH 5.0
- Recombinant Human MANBA (rhMANBA) (Catalog # 10520-GH)
- Substrate: 4-Methylumbelliferyl-beta -D-mannopyranoside (Sigma, Catalog # M0905), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhMANBA to 8 µg/mL in Assay Buffer.
- Dilute Substrate to 1 mM in Assay Buffer.
- Load 50 μL of 8 µg/mL rhMANBA into the plate, and start the reaction by adding 50 μL of 1 mM Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of 1 mM Substrate.
- Read at excitation and emission wavelengths of 365 nm and 445 nm, respectively, (top read) in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU) |
| amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard 4-Methylumbelliferone (Sigma, Catalog # M1381).
- rhMANBA: 0.4 µg
- Substrate: 0.5 mM
Scientific Data
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2 μg/lane of Recombinant Human MANBA Protein (Catalog # 10520-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 110 kDa.
Reconstitution Calculator
Background: MANBA/Beta-Mannosidase
MANBA is a lysosomal beta -mannosidase in the degradation pathway of N-glycans and catalyzes the removal of the innermost beta (1-4)-linked mannose residue (1). Mutations of MANBA can lead to cellular storage and secretion in the urine of the disaccharide, Man( beta 1–4)GlcNAc (2), and the symptoms of lysosomal storage disease (LSD) beta -mannosidosis, associated with mental retardation, behavioral problems including hyperactivity and aggressiveness, developmental delay, frequent infections, and hearing loss (3). Angiokeratoma corporis diffusum is another symptom of beta ‐mannosidosis that is characterized by involuntary eye movements (4) and can be found in other LSDs such as Fabry disease, beta ‐galactosidosis, fucosidosis, aspartylglucosaminuria, and galactosialidosis (5). LSDs are progressive disorders and are among the most common genetic disorders affecting children (6). Patients with LSDs are usually treated with enzyme replacement therapy (ERT) in which recombinant enzymes are administered to them for direct uptake into the lysosome (7). MAMBA belongs to the glycosyl hydrolase (GH) 2 family, in which enzymes are classified on the basis of sequence similarity (8). The only other human GH2 enzyme is beta ‐glucuronidase, deficiency of which leads to Sly syndrome (9). GH2 family members are characterized by a catalytic TIM barrel (10), in which the catalytic acid-base and nucleophile residues lie on beta ‐strands 4 and 7, respectively. Human beta ‐mannosidase is sensitive to pH with optima between 4.5 and 5.0 (11).
- Alkhayat, A. H. et al. (1998) Hum Molec Genet 7:75.
- Hokke, C.H. et al. (1990). J Inherit Metab Dis 13:273.
- Wenger, D.A. et al. (1986). N Engl J Med 315:1201.
- Gytz, H. et al. (2019) FEBS J. 286:1319.
- Rodriguez‐Serna, M. et al. (1996) Arch Dermatol 132:1219.
- Hawkins‐Salsbury, J.A. et al. (2011) Hum Mol Genet 20:R54.
- Wyatt, K. et al. (2012) Health Technol Assess 16:1.
- Cantarel, B.L. et al. (2009). Nucleic Acids Res 37:D233.
- Sly, W.S. et al. (1973). J. Pediatr. 82:249.
- Henrissat, B. et al. (1995). Proc Natl Acad Sci USA 92:7090.
- Percheron, F. et al. (1992). Biochimie 74:5.
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