Abstract
Gaussia luciferase (Gluc)—with its many favorable traits such as small size, bright emission, and exceptional stability—has become a prominent reporter protein for a wide range of bioluminescence-based detection applications. The ten internal cysteine residues crucial to functional structure formation, however, make expression of high quantities of soluble protein in bacterial systems difficult. In addition to this challenge, the current lack of structural data further complicates the use of Gluc for in vitro applications, such as biosensors, or cellular delivery, both of which rely heavily on robust and reproducible bioconjugation techniques. While Gluc is already appreciably small for a luciferase, a reduction in size that still retains significant bioluminescent activity, in conjunction with a more reproducible bioorthogonal method of chemical modification and facile expression in bacteria, would be very beneficial in biosensor design and cellular transport studies. We have developed truncated variants of Gluc, which maintain attractive bioluminescent features, and have characterized their spectral and kinetic properties. These variants were purified in high quantities from a bacterial system. Additionally, a C-terminal linker has been incorporated into these variants that can be used for reliable, specific modification through tyrosine-based bioconjugation techniques, which leave the sensitive network of cysteine residues undisturbed.
Highlights
In recent years, Gaussia luciferase (Gluc)—from the mesopelagic copepod Gaussia princeps—has emerged as a prominent reporter protein for bioluminescent detection applications
Gluc requires special attention to expression conditions for proper folding, and while it has been expressed in mammalian systems[1] it has proven difficult to express in high concentration from bacterial systems
Gluc was purified in high concentration from E. coli; 15–20 mg per 1 L culture was recovered from the soluble fraction by immobilized-metal affinity chromatography (IMAC) following cell lysis
Summary
Gaussia luciferase (Gluc)—from the mesopelagic copepod Gaussia princeps—has emerged as a prominent reporter protein for bioluminescent detection applications. The sequence for Gluc contains two structural domains exhibiting a high degree of homology (see Fig. 1)[4]. This is a common trait of marine luciferases, and is observed in the Metridia luciferase isoforms[5], which share ~60–70% sequence homology with Gluc. In 2009, Maguire et al created a variant of Gluc containing the single point mutation M43I, which provided glow-type kinetics in the presence of 0.1% Triton X-10011. This enhanced stability of the bioluminescent emission was favorable for streamlining high-throughput assays utilizing Gluc as a reporter. More recently in 2013, Degeling et al.[12] created a variant—L30S, L40P, M43V (Gluc4)—which demonstrated exceptional glow-type kinetics both in the presence and absence of Triton X-10012
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