Improved yellow-green split fluorescent proteins for protein labeling and signal amplification

The flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP(1-10/11) split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP(11) tags. To improve the FP(1-10/11) toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A(1-10/11) and mNG3K(1-10/11)) and one mClover-based split FP (CloGFP(1-10/11)). mNG3A(1-10/11) and mNG3K(1-10/11) not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG2(11) fragments in mammalian cells.