1. |
Authentic human acetylcholinesterase (AChE) was expressed inEscherichia coli under regulation of the constitutivedeo promoter or the thermoinducible PL promoter.
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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.
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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.
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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.
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5. |
The recombinant enzyme analogue was indistinguishable from native AChE isolated from erythrocytes in terms of substrate specificity and inhibitor selectivity.
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