Preparation of recombinant firefly luciferase by a simple and rapid expression and purification method and its application in bacterial detection

A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase, equipped with a polyhistidine affinity tag, was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108, and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein, the ATP content of bacteria was 9.48×10-16mol/mL, and was identical to the bacteria counts (4500CFU/mL) in order of magnitude. Taken together, our results provided a simple and efficacious method of the preparation of recombinant luciferase, which could be applied in the determination of bacteria via ATP bioluminescence.     

生物发光筛选技术

生物发光是指生命体通过多种化学发光系统发出可见光的能力。生物发光反应需要3种反应物：荧光素、荧光素酶和分子氧。荧光素酶可对其底物一荧光素的氧化反应进行催化，生成一种不稳定的中间体。在不稳定的中间体分解时就会发光并生成氧化荧光素。在一些反应中，可能会需要其它的反应物，包括钙、镁的阳离子和三磷酸腺苷（ATP）和亚硝酸还原酶（NAD（P）H）等。