Enzyme-complemented activatorsorbent assay (ECASA): genetic engineering for enzyme-linked immunosorbent assay-type mercuric ion detection.
The sensor component of bacterial mercury resistance systems is the metalloregulatory protein MerR, which has nanomolar sensitivity and high selectivity for Hg(II). A fusion protein of MerR and the alpha-peptide part of beta-galactosidase (LacZalpha) was constructed by fusing the relevant genes. The protein exhibited both MerR functions and alpha-complementing activity to the inactive LacZDeltaM15 (M15) protein. The bifunctional character of the appropriate MerR-LacZalpha-complemented M15 protein (MerR-LacZalpha:M15 protein complex) was used to develop a Hg(II)-specific enzyme-complemented activatorsorbent assay. Hg(II) was immobilized and presented on a matrix taking advantage of the high affinity of Hg(II) to SH residues. The immobilized Hg(II) could be specifically detected down to the parts-per-billion level by quantifying the beta-galactosidase activity of the bound fusion protein complex.
Klein
J
J
Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, Stuttgart, D-70569, Germany. kjoachim@gensun.biologie.uni-stuttgart.de
Mattes
R
R
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D004352:Drug Resistance, Microbial; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D005838:Genotype; D008628:Mercury / Q000032:analysis* / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000096:biosynthesis
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C051124:MerR protein, Bacteria; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D008628:Mercury