Luciferases are a class of enzymes that emit light as part of their catalytic function. Luciferases have been found widely distributed in nature and have in many cases been employed as reporter systems for biochemical studies. The focus of this work has been to optimize the affinity of the split-luciferase system NanoBiT, engineered by the Promega corporation from the basis of the luciferase of Oplophorus gracilirostris, a small deep-sea shrimp.
The NanoBiT system works by splitting the luciferase into an inactive, truncated luciferase, LgBiT, and an 11- amino acid peptide, SmBiT, which can restore luminescence function when binding to the LgBiT. This complementation can occur even when the peptide is fused to another protein. This makes it useful as a tag on other proteins, to detect and quantify them with a high degree of sensitivity and accuracy.
To increase the functionality of this system further, we wished to optimize the affinity of the peptide, by exploring its sequence space in more detail. This was done through the construction of an in vivo screening platform, to allow efficient luminescence screening of libraries of mutant variants of the SmBiT peptide.
In this process, libraries of variant peptides were screened for their ability to complement the truncated luciferase. From those results, analysis was done to estimate the contributions of the substitutions found, resulting in a prediction of specific candidate substitutions likely to improve function. The candidates were then validated in vitro, resulting in two improved peptides; one with improved affinity and another with improved luminescent activity.