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

作　　者：Qin Xiao1,2,Hui Chen2,Jin-Ming Lin2 1. College of Ocean,Hebei Agricultural University,Qinhuangdao 066003 2. The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology,Department of Chemistry,Tsinghua University,Beijing 100084,China. 

出　　处：《Journal of Pharmaceutical Analysis》2010年第2期97-101,共5页药物分析学报（英文版）

基　　金：supported by the National Key Technology R & D Program (2006BAK02A13);the National Basic Research Program of China (973Program, No.2007CB714507)

摘　　要：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.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.

关 键 词：ATP bioluminescence bacterial detection EXPRESSION firefly luciferase PURIFICATION 

分 类 号：Q78[生物学—分子生物学]