Expression and processing of vertebrate acetylcholinesterase in the yeast Pichia pastoris.

摘要: In the methylotrophic yeast Pichia pastoris, we expressed rat acetylcholinesterase H and T subunits (AChEH AChET respectively), as well truncated from (W553stop or AChETDelta, which most of T-peptide was removed) Bungarus (V536stop, AChENAT, AChEDelta, reduced to catalytic domain). We show that AChEH are processed into same molecular forms in vivo transfected mammalian cells, but lytic processes converting amphiphilic non-amphiphilic derivatives appear be more active yeast. The production glycophosphatidylinositol (GPI)-anchored molecules (dimers, with a small proportion monomers) demonstrates P. pastoris can correctly process C-terminal GPI-addition signal. Truncated AChE molecules, exclusively generated monomers, were released efficiently thus produced activity. hope increasing AChE, replaced endogenous signal peptide by prepeptides, without propeptide. found presence propeptide, does not exist prevent proper folding enzyme, it may either increase decrease yield secreted depending on peptide. Surprisingly, highest obtained For all combinations, 2-3 times higher for than probably reflecting differences efficiency stability polypeptides. Michaelis constant (Km), inhibition excess substrate (Kss) (kcat) values recombinant AChEs both COS essentially identical those corresponding natural enzymes, Ki active-site peripheral-site inhibitors (edrophonium, decamethonium, propidium) similar.