Abstract
Fluorescent proteins with varying colors are indispensable tools for the life sciences research community. These fluorophores are often developed for use in mammalian systems, with incremental enhancements or new versions published frequently. However, the successful application of these labels in other organisms in the tree of life, such as the fungus Candida albicans, can be difficult to achieve due to the difficulty in engineering constructs for good expression in these organisms. In this contribution, we present a palette of Candida-optimized fluorescent proteins ranging from cyan to red and assess their application potential. We also compare a range of reported expression optimization techniques, and find that none of these strategies is generally applicable, and that even very closely related proteins require the application of different strategies to achieve good expression. In addition to reporting new fluorescent protein variants for applications in Candida albicans, our work highlights the ongoing challenges in optimizing protein expression in heterologous systems.
Highlights
Fluorescent proteins with varying colors are indispensable tools for the life sciences research community
This is the case for the Cyan Fluorescent Protein (CFP), which gives a low overall fluorescent signal when used in C. albicans (WVG and LD, personal communications, 2018)
The codon usage table for C. albicans was already available on the OPTIMIZER server and applied to obtain the OPTIMIZER entire genome sequences (OPT Entire)
Summary
Fluorescent proteins with varying colors are indispensable tools for the life sciences research community. The successful application of these labels in other organisms in the tree of life, such as the fungus Candida albicans, can be difficult to achieve due to the difficulty in engineering constructs for good expression in these organisms In this contribution, we present a palette of Candida-optimized fluorescent proteins ranging from cyan to red and assess their application potential. We selected a rationally improved version of CFP called mTurquoise[2], which has a fluorescence quantum yield of 93% and improved photostability, for further codon optimization[15] Whilst these previous FPs are all related to the classical GFP isolated from Aequorea victoria, red fluorescent proteins (RFP) were isolated from Anthozoa species. Together with the advent of super-resolution microscopy methods, such as PALM17, RESOLFT18 and pcSOFI19, came the quest for novel and improved photoswitchable fluorescent proteins (PSFP) These proteins are able to reversibly switch between a dark ‘off ’ state and a bright ‘on’ state. One of these proteins is the green fast-forming (ff)Dronpa, which has proven suitable for sub-diffraction imaging[20]
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