A calibration curve for immobilized dihydrofolate reductase activity assay.

Priyanka Singh,Holly Morris,Amnon Kohen,Alexei V Tivanski

doi:10.1016/j.dib.2015.04.005

Abstract

An assay was developed for measuring the active-site concentration, activity, and thereby the catalytic turnover rate (kcat) of an immobilized dihydrofolate reductase model system (Singh et al., (2015), Anal. Biochem). This data article contains a calibration plot for the developed assay. In the calibration plot rate is plotted as a function of DHFR concentration and shows linear relationship. The concentration of immobilized enzyme was varied by using 5 different size mica chips. The dsDNA concentration was the same for all chips, assuming that the surface area of the mica chip dictates the resulting amount of bound enzyme (i.e. larger sized chip would have more bound DHFR). The activity and concentration of each chip was measured.

Highlights

A calibration curve for immobilized dihydrofolate reductase activity assay Priyanka Singh, Holly Morris, Alexei V
An assay was developed for measuring the active-site concentration, activity, and thereby the catalytic turnover rate of an immobilized dihydrofolate reductase model system (Singh et al, (2015), Anal
In the calibration plot rate is plotted as a function of DHFR concentration and shows linear relationship

Summary

Immobilization procedure

Escherichia coli DHFR was expressed, purified, and stored as discussed elsewhere [2,3]. The labeled (thiol/amino terminated) dsDNA linkers were generated by PCR using Taq DNA polymerase and a pEt22bDHFR vector as a template [3]. The labeled dsDNA linkers were attached to pre-activated mica using ethanolamine and 1,4-phenylene diisocyanate through the 50 amino terminated end as described elsewhere [4,5,6,7,8,9,10]. After activation of the dsDNA containing thiol group with tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) [11], the plate was submerged in activated DHFR in MTEN buffer (pH 7.5), resulting in disulfide bond formation between the thiol on DNA and a surface cysteine residue on the enzyme

Atomic force microscopy imaging

Calibration plot