Identification of a carboxylesterase expressed in protoplasts using fluorescence-activated cell sorting.
A method was developed to identify plant carboxylesterases using a homologous expression system with the capacity for high-throughput screening based on fluorescence-activated cell sorting (FACS). Protoplasts of Arabidopsis thaliana were prepared and transfected with a mutated (Cys59Ser) Arabidopsis S-formylglutathione hydrolase (atsfghm), which encoded a carboxylesterase highly active in the hydrolysis of the vital marker methylumbelliferyl acetate (MUA) to the fluorophore methylumbelliferone (MU). Unlike all other Arabidopsis carboxylesterases studied to date, AtSFGH and its more stable mutant variant AtSFGHm are insensitive to inhibition by organophosphate insecticides, such as paraoxon. By making use of the combined traits of a high carboxylesterase activity towards MUA and a lack of sensitivity to paraoxon, FACS was employed to selectively collect catalytically active atsfghm-transformed protoplasts. A population of 400,000 protoplasts containing 8000 sfghm transformants was treated with paraoxon to inhibit endogenous esterase activity and then fed with MUA. Fluorescent cells expressing the AtSFGHm enzyme were then collected by FACS, and the presence of the respective transgene was confirmed by polymerase chain reaction, with 9.6% of the transformants recovered. We suggest that the use of FACS to identify other carboxylesterases which can be catalytically determined using plant cell fluorescence-based assays could be a powerful method for the high-throughput screening of new enzymes, especially those which do not express well in microbial hosts.
Cummins
Ian
I
Centre for Bioactive Chemistry, Durham University, Durham DH1 3LE, UK.
Steel
Patrick G
PG
Edwards
Robert
R
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D043182:Carboxylesterase / Q000235:genetics* / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods*; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D010261:Paraoxon / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011523:Protoplasts / Q000201:enzymology; D014170:Transformation, Genetic
D002800:Cholinesterase Inhibitors; D010940:Plant Proteins; D049452:Green Fluorescent Proteins; D006923:Hymecromone; D043182:Carboxylesterase; D010261:Paraoxon