Design of a coupled bioluminescent assay for a recombinant pyruvate kinase from a thermophilic Geobacillus.

A simple and rapid method using coupled bioluminescent assay was developed to determine level of ADP. ADP is involved in many biological reactions and ADP assay can be used for assaying some reactions universally by monitoring ADP formation or depletion. ADP analysis involves incubation of ADP or extracts containing ADP with pyruvate kinase (PK) and PEP. The ATP formed by this reaction is determined by measuring the intensity of the initial light flash produced when luciferin-luciferase preparation injected into the reaction mixture. In regard to the main role of the PK in this assay, the gene of PK from a Geobacillus species has been cloned in expression vector pET28a (+), sequenced and overexpressed in Escherichia coli. Recombinant protein was purified using Ni-NTA column and then the purified PK was used in a coupled bioluminescent assay for ADP measurement. Kinetic properties of PK are determined according to a bioluminescent assay using firefly luciferase.