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

• Main Authors: Lundqvist, Magnus, Thalén, Niklas, Volk, Anna-Luisa, Hansen, Henning Gram, von Otter, Eric, Nygren, Per-Åke, Uhlen, Mathias, Rockberg, Johan
• Other Authors: School of Biological Sciences
• Format: Article
• Language: English
• Published: 2019
• Subjects: DRNTU::Science::Biological sciences; Chromophore; Protein Secretion
• Online Access: https://hdl.handle.net/10356/85425; http://hdl.handle.net/10220/48210
• Institution: Nanyang Technological University

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.