Recombinant human acetylcholinesterase expressed in Escherichia coli: refolding, purification and characterization.

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.