Ryohei Sato

Expression of carp acetylcholinesterase gene in yeast

Ryo-hei YAMADA, Yoshio KERA, Shouji TAKAHASHI

Organophosphorus and carbamate compounds have been used in large amounts as pesticides. Because the compounds irreversibly inhibit acetylcholinesterase ( AChE ), the tissue levels of AChE activity in various fishes have been used as a potential biomarker for exposure to these compounds, but the methods so far employed have not given reliable results. We therefore attempt to develop a new method using the immunological specific activity of AChE of common carp ( Cyprinus carpio ). To obtain pure AChE required for raising antibodies against the enzyme, we cloned the carp AChE gene and overexpressed it in the yeast.
We cloned the carp AChE gene and constructed three different yeast expression vectors. The vectors were introduced into the methylotrophic yeast Pichia pastoris. Semi-quantitative PCR and Southern blot analysis showed that the AChE gene was present as a single copy in the genome of each transformant. In all the transformants, we succeeded in producing the carp AChE in the culture medium. At present, maximum AChE activity ( 5930 nmol/min/ml ) was observed in the culture medium of one of the transformants after cultivation for 10 days at 15��. To produce more AChE in the medium, optimization of the culture conditions is in progress.