| Autor: |
Schwarzmann, G, Klima, H, Klein, A, van Echten, G, Sandhoff, K, Suzuki, K |
| Jazyk: |
angličtina |
| Rok vydání: |
1993 |
| Předmět: |
|
| DOI: |
10.17615/p721-1g03 |
| Popis: |
The cDNA of the human GM2-activator protein was cloned into the expression vector pHX17. The plasmid encodes a fusion protein with a hexahistidine tail and a Factor Xa cleavage site at its N-terminus. The recombinant protein was purified from cell homogenates under denaturing conditions by metal-ion affinity chromatography in a single step and then was refolded. The hexahistidine tail could be removed when desired by digestion with Factor Xa. In a functional assay, the GM2-activator thus generated from Escherichia coli and renatured, with or without the hexahistidine tail, was as active as the native GM2-activator protein that was purified from human tissue. When added to the culture medium, the recombinant carbohydrate-free GM2-activator, carrying the hexahistidine tail, could be taken up efficiently and restored the degradation of ganglioside GM2 to normal rates in mutant fibroblasts with the AB variant of GM2-gangliosidosis, which is characterized by a genetic defect in the GM2-activator protein. The prokaryotic expression system is useful for producing milligram quantities of a pure and functionally active GM2-activator. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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