Dihydrofolate reductase (DHFR) reporter enzyme assay for Haloferax volcanii

Abstract The dihydrofolate reductase (DHFR) is routinely used a reporter enzyme for H. volcanii. The DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate and the concomitant oxidation of NADPH to NADP+. This leads to a reduction of extinction at 340 nm, which is measured to quantify the DHFR activity. To avoid background, it is best to use an H. volcanii strain with a deletion of the chromosomal dhfr gene, which is available upon request (soppa@bio.uni-frankfurt.de). However, the expression level of the chromosomal dhfr gene is very low, so that it is also possible to use the wildtype strain and subtract the DHFR background level. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the dhfr gene in a translational fusion with the gene of interest.