Mutation Report for: C580S_human-ACHE

A large-scale preparation of a recombinant human acetylcholinesterase (rhAChE) mutant harbouring a CyS580-->Ser substitution, expressed in Escherichia coli, was refolded following solubilization of the inclusion bodies. Refolded active rhAChE was purified by DEAE-Sepharose and affinity chromatography to apparent homogeneity with a specific activity (4572 units/mg) similar to that of erythrocyte AChE. The stability of the purified enzyme at 22-37 degrees C was dependent on the presence of 0.5 mg/ml BSA, and the optimum pH for stability was 9.0. rhAChE has a UV-absorbance spectrum typical of a tryptophan-rich protein, with a distinct shoulder at 290 nm and a high absorption coefficient at 280 nm (epsilon 1% = 23.1). The tryptophan residues in active rhAChE are located in an apolar environment, characteristic of a globular molecule. The difference in amino acid composition between red-blood-cell-derived and recombinant hAChE is probably reflected in their different pI values, namely 5.5-5.8 and 4.6-5.2 respectively. The CD spectrum of rhAChE is typical for an alpha/beta protein, indicating 39% alpha-helix and 22% beta-sheet. This secondary structure is similar to that determined for the Torpedo (electric fish) AChE, by both CD and X-ray crystallography. On the other hand, a purified misfolded and inactive molecule displays a decrease in alpha-helical content to 24%, accompanied by an increase in beta-sheet up to 42%, indicative of extensive changes in the conformation of the protein. On the whole, the recombinant enzyme has been refolded into a native-like conformation possessing full activity, and is thus similar to the naturally occurring red-blood-cell-derived hAChE.

1. Authentic human acetylcholinesterase (AChE) was expressed in Escherichia coli under regulation of the constitutive deo promoter or the thermo-inducible lambda PL 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.