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.