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Updated: Apr 21, 2026
A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions
Published on: June 6, 2018
Quantitation of CYP24A1 enzymatic activity with a simple two-hybrid system
Amy Mugg1, Balazs Legeza, Meng Kian Tee
1Department of Pediatrics, University of California, San Francisco, San Francisco, California 94143-1346.
Context:
Mutations of the CYP24A1 gene encoding the 24-hydroxylase (24OHase) that inactivates metabolites of vitamin D can cause hypercalcemia in infants and adults; in vitro assays of 24OHase activity have been difficult.
Objective:
We sought an alternative assay to characterize a CYP24A1 mutation in a young adult with bilateral nephrolithiasis and hypercalcemia associated with ingestion of excess vitamin D supplements and robust dairy intake for 5 years.
Methods:
CYP24A1 exons were sequenced from leukocyte DNA. Wild-type and mutant CYP24A1 cDNAs were expressed in JEG-3 cells, and 24OHase activity was assayed by a two-hybrid system.
Results:
The CYP24A1 missense mutation L409S was found on only one allele; no other mutation was found in exons or in at least 30 bp of each intron/exon junction. Based on assays of endogenous 24OHase activity and of activity from a transiently transfected CYP24A1 cDNA expression vector, JEG-3 cells were chosen over HepG2, Y1, MA10, and NCI-H295A cells for two-hybrid assays of 24OHase activity. The apparent Michaelis constant, Km(app), was 9.0 ± 2.0 nM for CYP24A1 and 8.6 ± 2.2 nM for its mutant; the apparent maximum velocity, Vmax(app), was 0.71 ± 0.055 d(-1) for the wild type and 0.22 ± 0.026 d(-1) for the mutant. As assessed by Vmax/Km, the L409S mutant has 32% of wild-type activity (P = .0012).
Conclusions:
The two-hybrid system in JEG-3 cells provides a simple, sensitive, quantitative assay of 24OHase activity. Heterozygous mutation of CYP24A1 may cause hypercalcemia in the setting of excessive vitamin D intake, but it is also possible that the patient had another, unidentified CYP24A1 mutation on the other allele.
