Production of Enzymatically Active Human Acetylcholinesterase in E. Coli

Abstract : Human acetylcholinesterase (hAChE) was expressed in Escherichia coli under regulation of the constitutive deo promoter or the thermoinducible lambda P sub L promoter. To facilitate the expression in the prokaryotic system, the recombinant human AChE (rhAChE) cDNA was modified at the N-terminus, by site- directed mutagenesis, in order to replace some of the guanisine and cytosine (GC)-rich regions by adenine thimidine (AT). These modifications did not alter the amino acid sequence but resulted in ample production of the protein. 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. Active rhAChE was obtained after solubilization, folding and oxidation; however, the overall yield of the active enzyme was low. A 20- to 40-fold increase in the process yield of active rhAChE activity was achieved by replacing Cys(580) by Ser. Substrate specificity and inhibitor selectivity of the recombinant mutant enzyme harboring Ser at amino acid number 580 were indistinguishable from those of the native AChE isolated from human erythrocytes.