Production of Enzymatically Active Human Acetylcholinesterase in E. Coli
Final rept. 1 Sep 1990-31 Aug 1993
BIOTECHNOLOGY GENERAL CORP NEW YORK
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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 Cys580 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.