Biotechnology Letters 23: 417–422, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 417 Production of Drosophila melanogaster acetylcholinesterase in Saccharomyces cerevisiae K.C.A. Stopps, B.P.G. Curran, S.A. Khalawan & C.A. Malcolm ∗ School of Biological Sciences, Queen Mary and Westfield College, University of London, Mile End Road, London, E1 4NS, UK ∗ Author for correspondence (Fax: +44-20-8983-0973; Email: c.a.malcolm@qmw.ac.uk) Received 13 November 2000; Revisions requested 22 November 2000; Revisions received 5 January 2001; Accepted 8 January 2001 Key words: acetylcholinesterase, Drosophila melanogaster, Saccharomyces cerevisiae Abstract Biologically active acetylcholinesterase from Drosophila melanogaster was constitutively expressed from a high copy number plasmid in Saccharomyces cerevisiae. Cellular metabolism and plasmid copy number were unaffected by expression. Based on comparative PMSF inhibition, the K cat value (6430 mol s −1 ) was commensurate with human and Torpedo acetylcholinesterases. A 1.7 knt truncated mRNA transcript was observed on Northern blot analysis. Introduction Acetylcholinesterase (AChE) plays a critical role in regulation of the neurotransmitter acetylcholine and is the focus of studies in a diversity of areas, ranging from insecticide resistance in insect pests (Malcolm et al. 1998) to Alzheimer and Parkinson diseases in humans (Shinotoh et al. 2000). The vast majority of commercially available AChE is currently extracted from animal tissues, making the development of suitable genetically engineered sys- tems highly desirable. Such procedures can be used to generate novel variants of the enzyme and to provide unlimited supplies of AChE from any source for which a cDNA is available. AChE has already been heterolo- gously expressed using procedures involving various diverse species including a yeast, Pichia pastoris, (Morel & Massoulie 1997), yet surprisingly, previous attempts to obtain AChE production in Saccharomyces cerevisiae have failed. We report here expression of biologically active Drosophila melanogaster AChE in S. cerevisiae strain BJ2168 and note that poor stability of the mRNA tran- script appears to be a contributing factor to previous difficulties in attaining this goal. Materials and methods Biological materials Saccharomyces cerevisiae strain BJ2168 (Mat a, leu 2, trp 1, ura 3-52, prb 1-1122, pep 4-3, prc 1-407 gal 2) was obtained from the Yeast Genetics Stock Center (MCB/Biophysics and Cell Physiology, 102 Donner Laboratory, University of California, Berkeley, CA 94720). Escherichia coli strain DH5α was from Gibco BRL Life Technologies. Yeast and bacterial strains were propagated on standard media (Guthrie & Fink 1991). pKS-DAChE is a recombinant Bluescript KS+ (Stratagene) plasmid carrying the Drosophila melano- gaster Ace cDNA with the leader sequence truncated so that the first ATG is the correct one (Hall & Spencer, 1986). pG3 is a 9.1 kb vector for high level constitutive expression in S. cerevisiae using the glyceraldehyde 3-phosphate dehydrogenase promoter. It was prepared from pUC18 plasmid and sections of yeast DNA, in- cluding the TRP1 gene and a segment of 2-μm DNA (see Guthrie & Fink 1991). pDCD112 is a recombi- nant pUC18 plasmid containing yeast ribosomal RNA gene (rDNA) and 2-μm DNA (Row 1993). Purified Torpedo marmorata AChE was bought from Sigma.