Low levels of endogenous cholinesterases support the choice of cows, sheep and goats for the transgenic expression of human butyrylcholinesterase in milk

Abstract

Butyrylcholinesterase purified from human plasma (Hu BChE) as well as recombinant (r) Hu BChE are candidate enzymes that can protect humans from toxicity of organophosphorus compounds (OPs). Domestic animals such as cows, pigs, sheep, and goats have been used for the transgenic expression of a variety of valuable therapeutic proteins. Indeed, rHu BChE was successfully expressed in the milk of transgenic goats, but the presence of any endogenous cholinesterases (ChE) in milk would interfere with the isolation of expressed rHu BChE. The aim of this study was to determine the presence of endogenous ChEs in bovine, ovine, caprine, and porcine milk to determine the suitability of these species for the production of rHu BChE. Using acetyl- and butyryl- thiocholine as substrates, ChE activity (2–4 U/mL) was detected in pig milk only. ChE activities in milk from other animals were <0.01 U/mL and could only be detected following enrichment on procainamide-Sepharose gel. Two different methods based on measuring activity in the presence of acetylcholinesterase (AChE)- or BChE- specific inhibitors were used to estimate the proportions of AChE and BChE activities in enriched milk. Monoclonal antibodies (MAbs), against fetal bovine serum AChE that recognize AChEs from ruminants only, were also used to confirm the identity of AChEs. While bovine and ovine milk contain both AChE and BChE activities, caprine and porcine milk contain predominantly BChE activity. The presence of very low ChE activity supports the choice of cows, sheep, and goats for the transgenic expression of rHu BChE in milk.