Acetylcholinesterase assay for rapid expression screening in liquid and solid media.
The synaptic enzyme acetylcholinesterase (AChE), which is the target of many insecticides and potential warfare agents, is implied in Alzheimer's disease and is a good potential candidate to be used in biosensors. This promotes a strong demand for production of recombinant AChE to be used in various studies. A promising expression system is the yeast Pichia pastoris, but the expression efficiency needs to be improved. Optimization studies require a rapid and efficient screening test to detect positive yeast colonies after transformation. Using indoxylacetate as a substrate, we designed a chromogenic test that is not interfered with by the culture media background color and, thus, is suitable for microplate screening. Moreover, it was possible to adapt the test for direct on-plate detection of AChE-expressing colonies.
Villatte
F
F
Stuttgart University, Stuttgart, Germany.
Bachman
T T
TT
Hussein
A S
AS
Schmid
R D
RD
D000110:Acetylcholinesterase / Q000235:genetics* / Q000378:metabolism; D000122:Acetylthiocholine / Q000378:metabolism; D000818:Animals; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007211:Indoles / Q000378:metabolism; D009559:Nippostrongylus / Q000201:enzymology; D010843:Pichia / Q000201:enzymology / Q000235:genetics
D017077:Culture Media, Conditioned; D007211:Indoles; D000122:Acetylthiocholine; C053640:indoxyl acetate; D004228:Dithionitrobenzoic Acid; D000110:Acetylcholinesterase