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Cloning and functional expression of a cDNA encoding coffee bean alpha-galactosidase
1Lindsley F. Kimball Research Institute, New York Blood Center, New York 10021.
Gene
|March 25, 1994
Summary
Researchers isolated and sequenced the coffee bean alpha-galactosidase (alpha Gal) gene. The resulting protein demonstrated enzymatic activity, confirming its function in removing alpha-galactose residues.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Coffee bean alpha-galactosidase (alpha Gal) is utilized for modifying red blood cell surface glyco-conjugates.
- This enzyme aids in studies of blood group conversion by removing terminal alpha-galactose residues.
Purpose of the Study:
- To isolate and sequence the full-length clone of coffee bean alpha-galactosidase.
- To confirm the authenticity and enzymatic activity of the cloned alpha Gal protein.
Main Methods:
- Polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques were employed for gene isolation.
- The full-length cDNA clone (1.4 kb) was sequenced, encoding a 378 amino acid protein.
- Authenticity was verified through sequence alignment and antibody-based immune reactions.
Main Results:
- The complete coding sequence for coffee bean alpha Gal was successfully obtained.
- The deduced amino acid sequence perfectly matched the purified enzyme.
- Recombinant protein produced in insect cells exhibited enzymatic activity against a synthetic substrate.
Conclusions:
- The study successfully isolated and characterized the coffee bean alpha Gal gene and its protein product.
- The findings confirm the enzyme's structure and function, paving the way for further applications in glycobiology and blood group research.