Abstract
The flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP11 tags. To improve the FP1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A1-10/11 and mNG3K1-10/11) and one mClover-based split FP (CloGFP1-10/11). mNG3A1-10/11 and mNG3K1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG211 fragments in mammalian cells.
Highlights
Fluorescent proteins (FPs), such as Green Fluorescent Protein (GFP), are a group of structurally homologous proteins that are widely used as genetically encoded fluorescent tags
After four rounds of directed evolution and one round of DNA shuffling, the brightness of E. coli colonies grown on Luria broth (LB) plates increased substantially (Fig 1B)
We used directed evolution combined with rational design to improve the brightness of two yellow-green split-FPs
Summary
Fluorescent proteins (FPs), such as Green Fluorescent Protein (GFP), are a group of structurally homologous proteins that are widely used as genetically encoded fluorescent tags. Their structure usually consists of a cylindrical β-barrel, comprised of 11 β-strands, and a chromophore formed within the β-barrel. Splitting the β-barrel between strand 10 and 11 produces a spontaneously self-associating split fluorescent protein system, which we named as FP1-10/11 [1]. The self-complementing nature of FP1-10/11 systems has enabled a wide range of applications, including detection of protein expression [1], visualization of cell-cell interactions [3, 5], and monitoring intracellular infection [6, 7].
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