Expression and reconstitution of biologically active human acetylcholinesterase fromEscherichia coli
| Autor: | Marian Gorecki, Meir Fischer, Ilana Liefer, Avner Ittah |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Recombinant Fusion Proteins
Genetic Vectors Molecular Sequence Data Biology medicine.disease_cause Inclusion bodies Substrate Specificity law.invention Serine Cellular and Molecular Neuroscience chemistry.chemical_compound law Complementary DNA Escherichia coli medicine Humans Promoter Regions Genetic Peptide sequence chemistry.chemical_classification Base Sequence Cell Biology General Medicine Acetylcholinesterase Molecular biology Enzyme chemistry Biochemistry Recombinant DNA Cholinesterase Inhibitors |
| Zdroj: | Cellular and Molecular Neurobiology. 13:25-38 |
| ISSN: | 1573-6830 0272-4340 |
| Popis: | 1. Authentic human acetylcholinesterase (AChE) was expressed inEscherichia coli under regulation of the constitutivedeo promoter or the thermoinducibleλPL promoter. 2. To facilitate expression in the prokaryotic system, recombinant human AChE (rhAChE) cDNA was modified at the N terminus by oligonucleotide substitutions in order to replace some of the GC-rich regions by AT. These modifications did not alter the amino acid sequence but resulted in ample production of the protein. 3. rhAChE accumulated in the cells and reached a level of 10% of total bacterial proteins. A partially purified inactive recombinant protein was recovered from inclusion bodies. 4. Active rhAChE was obtained after solubilization, folding, and oxidation, although the recovery of the active enzyme was low. A 20- to 40-fold increase in enzymatically active rhAChE was achieved by replacing Cys580 by serine. 5. The recombinant enzyme analogue was indistinguishable from native AChE isolated from erythrocytes in terms of substrate specificity and inhibitor selectivity. |
| Databáze: | OpenAIRE |
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