Optimized application of the secreted Nano-luciferase reporter system using an affinity purification strategy.

Jingzhe Li,Juying Zhong,Takashi Sato,Zhilan Guo,Yan Lei,Bo Yuan,Peng Huang,Yi Wang,Mengmeng Jin,Yanyan Ma,Dan Qian,Luyang Che,Changzhen Liu,Wataru Nishimura

doi:10.1371/journal.pone.0196617

Abstract

Secreted Nano-luciferase (secNluc) is a newly engineered secreted luciferase that possesses advantages of high structural stability, long half-life, and glow-type kinetics together with high light emission intensity, and thus would become one of the most valuable tools for bioluminescence assays. However, like other secreted luciferases, secNluc has to mix with the components in the conditioned medium surrounding test cells, or in the biological samples such as blood or urine after being secreted. These components may interfere with secNluc-catalyzed bioluminescence reactions and thus limit the application of the secNluc reporter system. In this study, we first examined the effects of three factors, pH, serum and residual reagents, on secNluc-catalyzed bioluminescence reactions, finding that these factors could interfere with bioluminescence reactions and result in background signal. To resolve these problems, we applied a simple affinity purification strategy in which secNluc was fused with a FLAG-tag, and anti-FLAG magnetic beads were used to catch and transfer the fusion protein to PBST, an optimal buffer for secNluc-catalyzed bioluminescence reactions that was identified in this study. The results indicated that this strategy could not only negate the interferences from serum or residual reagents and enhance the stability of light emission but also greatly increase signal intensity through enzyme enrichment. This strategy may contribute to biomedical studies that utilize secNluc and other secreted luciferases, especially those requiring superior sensitivity, low background noise and high reproducibility.

Highlights

Luciferases have been widely used as convenient and powerful reporting tools in numerous biomedical researches [1,2]
As a newly developed secreted luciferase, Secreted Nano-luciferase (secNluc) is thought to own the advantages of high structural stability, long half-life, and glow-type kinetics over other secreted luciferases including the Gaussia, Metridia and Cypridina luciferases[13]
The results showed that relative light unit (RLU) had no significant difference even though FLAG-tag existed or not at each pH value (Fig 3I), demonstrating that secNluc-catalyzed bioluminescence reactions was not impacted by FLAG-tag under different pH value

Summary

Introduction

Luciferases have been widely used as convenient and powerful reporting tools in numerous biomedical researches [1,2]. Nano-luciferase is a newly engineered luciferase enzyme that originates from the deep-sea shrimp Oplophorus gracilirostris and was structural optimized by Promega [12] This enzyme can be used as an intracellular reporter or as a secreted reporter when it is appended with a secretion signal from human IL6 [12]. This engineered luciferase enzyme is monomeric and maintains high enzyme stability both inside living cells and in culture medium, with several days of half-life[12,13]. The luminescent signal produced by Nanoluciferase is approximately 150-fold brighter than those produced by firefly or Renilla luciferases, which have been extensively applied in biomedical studies[13] These advantages make Nano-luciferase an attractive tool as a reporter for bioluminescence assays. Nano-luciferase has already been used to generate a wide variety of transgenic organisms and cells for in vivo and in vitro studies [14,15,16,17,18]

Methods

Results

Conclusion