RESUMO
Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.
Assuntos
Proteínas de Artrópodes , Crustáceos/genética , Luciferases , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/isolamento & purificação , Crustáceos/enzimologia , Luciferases/biossíntese , Luciferases/química , Luciferases/genética , Luciferases/isolamento & purificação , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , SolubilidadeRESUMO
Secreted reporter proteins are reliable modalities for monitoring of different biological processes, which can be measured longitudinally in conditioned medium of cultured cells or body fluids such as blood and urine, ex vivo. In this chapter, we will explore established secreted reporters and their applications and limitations for monitoring of promoter function. We will also describe both cell-based and blood-based assays for detecting three commonly used reporters: secreted alkaline phosphatase (SEAP ), Gaussia luciferase (Gluc), and Vargula luciferase (Vluc).