Summary
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1.
Authentic human acetylcholinesterase (AChE) was expressed inEscherichia coli under regulation of the constitutivedeo promoter or the thermoinducibleλPL 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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Fischer, M., Ittah, A., Liefer, I. et al. Expression and reconstitution of biologically active human acetylcholinesterase fromEscherichia coli . Cell Mol Neurobiol 13, 25–38 (1993). https://doi.org/10.1007/BF00712987
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DOI: https://doi.org/10.1007/BF00712987