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.

Study of the Binding Mode of Quaternary Ammonium Cationic Surfactant to Firefly Luciferase and the Prediction of Binary Mixture Toxicity

The wide use of quaternary ammonium cationic surfactants(QACs)results in their release into the environment.Most surfactants have significant biotoxicity.However,existing toxicity data on QACs are still lacking,especially regarding the joint toxic effects of their mixtures.In computer simulation technology,molecular docking technology is commonly used for studying the mode of action of receptors docking with ligands.The research of QACs mixture interaction is relatively rare,and the binding mode of QACs is unknown.In this study,molecular docking technology was applied to explore the QAC binding mode,and the concentration addition(CA)and independent action(IA)models were applied for predicting the mixture toxicity.Firefly luciferase(FLuc)was used as a macromolecular receptor,and five typical QACs:benzalkonium bromide(BLB),tetraethylammonium bromide(TLB),N,N,N-trimethyl-1-tetradecyl ammonium bromide(CTE),tetrabutylammonium chloride(TAC),and dodecyltrimethylammonium chloride(DTC)were used as small molecule ligands.Molecular docking technology was used to investigate the binding mode of macromolecules and small molecules.The luminescence inhibitory effects of individual compounds and binary mixture on FLuc were determined by microplate toxicity assay of luciferase.The prediction of mixture toxicity was performed by CA and IA.The results showed that the relative toxicity follows:TLB<TAC<DTC<BLB<CTE.TLB and TAC showed the BS-Ⅱbinding mode,and BLB,CTE and DTC showed the BS-Ⅲbinding mode.The toxicity of compounds with binding mode BS-Ⅱwas less than that of those with BS-Ⅲbinding mode.Not all mixtures with the same binding mode could be predicted by CA model,and the IA model did not effectively predict the toxicity of mixtures with compound with different binding modes.The mixture toxicities of QACs with the same binding mode mostly presented additive and synergistic effects,while the mixture toxic effects of QACs with different binding modes presented additive or antagonistic effects.

Myeloid zinc finger 1(MZF1) is the most important transcriptional factor for porcine follistatin promoter

Follistatin （FS） is a secreted protein, which was originally isolated from porcine follicular fluid. Expression of follistatin is tightly regulated during porcine growth and development. To study the essential transcriptional regions of the porcine FS promoter, ten primer pairs were designed to amplify segments with different lengths of the FS promoter from -1 800 to ＋16 bp. The products were then inserted into the pGL3-basic vector to analyze the relative luciferase activity. The results showed that the most remarkable changes of promoter activity were observed between constructs （-302/＋16 bp）-FS and （-180/＋16 bp）-FS （P〈0.01）. Further research showed that the reconstructed reporter plasmid lacking myeloid zinc finger 1 （MZF1） binding sequence had significantly decreased luciferase activity （P〈0.05）. Furthermore, the FS protein expression was significantly increased in PK15 cells while the MZF1 was overexpressed, suggesting that the short sequence ＂TCCCCACC＂ （the recognition site of transcription factor MZF1） was the most important for FS transcription activation in the porcine.