RECOMBINANT HUMAN ACETYLCHOLINESTERASE EXPRESSED IN ESCHERICHIA-COLI - REFOLDING, PURIFICATION AND CHARACTERIZATION

A large-scale preparation of a recombinant human acetylcholinesterase (rhAChE) mutant harbouring a Cys(580)-->Ser substitution, expressed in Escherichia coli, was refolded following solubilization of the inclus ion bodies. Refolded active rhAChE was purified by DEAE-Sepharose and affinity chromatography to apparent homogeneity with a specific activi ty (4572 units/mg) similar to that of erythrocyte AChE. The stability of the purified enzyme at 22-37 degrees C was dependent on the presenc e of 0.5 mg/ml BSA, and the optimum pH for stability was 9.0. rhAChE h as a UV-absorbance spectrum typical of a tryptophan-rich protein, with a distinct shoulder at 290 nm and a high absorption coefficient at 28 0 nn (epsilon(1%) = 23.1). The tryptophan residues in active rhAChE ar e located in an apolar environment, characteristic of a globular molec ule. The difference in amino acid composition between red-blood-cell-d erived 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-hel ix and 22% beta-sheet. This secondary structure is similar to that det ermined for the Torpedo (electric fish) AChE, by both CD and X-ray cry stallography. On the other hand, a purified misfolded and inactive mol ecule displays a decrease in alpha-helical content to 24%, accompanied by an increase in beta-sheet up to 42%; indicative of extensive chang es in the conformation of the protein. On the whole, the recombinant e nzyme has been refolded into a native-like conformation possessing ful l activity, and is thus similar to the naturally occurring red-blood-c ell-derived hAChE.