Expression and reconstitution of biologically active human acetylcholinesterase from Escherichia coli.
1. Authentic human acetylcholinesterase (AChE) was expressed in Escherichia coli under regulation of the constitutive deo promoter or the thermo-inducible lambda PL promoter. 2. To facilitate expression in the prokaryotic system, recombinant human AChE (rhAChE) cDNA was modified at the N terminus by oligonucleotide substitutions in order to replace some of the GC-rich regions by AT. These modifications did not alter the amino acid sequence but resulted in ample production of the protein. 3. 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. 4. Active rhAChE was obtained after solubilization, folding, and oxidation, although the recovery of the active enzyme was low. A 20- to 40-fold increase in enzymatically active rhAChE was achieved by replacing Cys580 by serine. 5. The recombinant enzyme analogue was indistinguishable from native AChE isolated from erythrocytes in terms of substrate specificity and inhibitor selectivity.
Fischer
M
M
Bio-Technology General Ltd., Kiryat Weizmann, Rehovot, Israel.
Ittah
A
A
Liefer
I
I
Gorecki
M
M
D000110:Acetylcholinesterase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity
D002800:Cholinesterase Inhibitors; D011993:Recombinant Fusion Proteins; D000110:Acetylcholinesterase