Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion

Abstract

Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1–10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1–10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1–10 in analyses. The pre-maturated GFP 1–10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1–10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1–10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

“…Dissociation of split-GFP fragments can only be achieved in vitro by photodissociation of the reconstituted barrel [112] or chemical disruption of peptide bonds in denaturation conditions [113]. Renaturation of isolated split-GFP fragments induces the reassociation of the two fragments into a functional GFP molecule, with a five-time increase in fluorescence intensity [114,115]. Following these observations, several studies have exploited the prematuration of the chromophore in order to improve split-GFP kinetics.…”

“…These include the production of prematured version of GFP1–10 or GFP1–9 by incorporating a proteolytic site in the GFP scaffold that release strand β11 [113,114] or β10–11 [116]. Alternative approaches involve coexpressing both fragments and inducing their disassembly under denaturation conditions [114], or purifying each fragment separately under acid-pH conditions [115].…”

Engineering of a Fluorescent Protein for a Sensing of an Intrinsically Disordered Protein through Transition in the Chromophore State

“…Although Aβ is one of the best-known IDPs, its sensing and analysis have been a challenge due to its tiny size and structural plasticity. , Due to the unstable structural characteristics of IDPs, rapid signal response is essential for their accurate detection. Typical β-strand-truncated GFPs are known to undergo chromophore maturation after the complementation, requiring a lengthy duration of time. , In contrast, β-strand-truncated GFPs with matured chromophore frequently exhibit a high background fluorescence, resulting in low signal-to-noise ratios, even in the absence of a complementing peptide. , Such truncated GFPs are known to retain partial structure and prone to significant photoexcitation via anionic chromophores. − …”

“…23,25 In contrast, β-strandtruncated GFPs with matured chromophore frequently exhibit a high background fluorescence, resulting in low signal-to-noise ratios, even in the absence of a complementing peptide. 26,27 Such truncated GFPs are known to retain partial structure and prone to significant photoexcitation via anionic chromophores. 28−30 To address these issues, we intended to develop ratiometric fluorescence sensing of Aβ instead of a traditional singlewavelength fluorescence measurement.…”

Substitutional landscape of a split fluorescent protein fragment using high-density peptide microarrays

“…By incubating a microarray library with an immature LOO-FP, one could study what the sequence requirements would be for both binding and chromophore maturation. Indeed, β-strand-assisted chromophore maturation of LOO11-GFP on solid support has already been demonstrated [ 24 ].…”