Engineering an efficient and bright split Corynactis californica green fluorescent protein.

Split green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein-protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale labeling of proteins in cells using CRISPR, but sets of efficient split fluorescent proteins that do not cross-react are needed for multiplexing experiments. We present a new monomeric split green fluorescent protein (ccGFP) engineered from a tetrameric GFP found in Corynactis californica, a bright red colonial anthozoan similar to sea anemones and scleractinian stony corals. Split ccGFP from C. californica complements up to threefold faster compared to the original Aequorea victoria split GFP and enable multiplexed labeling with existing A. victoria split YFP and CFP.

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

Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite ¼ split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein-protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.

Optimization of sample preparation and green color imaging using the mNeonGreen fluorescent protein in bacterial cells for photoactivated localization microscopy.

mNeonGreen fluorescent protein is capable of photo-switching, hence in principle applicable for super-resolution imaging. However, difficult-to-control blinking kinetics that lead to simultaneous emission of multiple nearby mNeonGreen molecules impedes its use for PALM. Here, we determined the on- and off- switching rate and the influence of illumination power on the simultaneous emission. Increasing illumination power reduces the probability of simultaneous emission, but not enough to generate high quality PALM images. Therefore, we introduce a simple data post-processing step that uses temporal and spatial information of molecule localizations to further reduce artifacts arising from simultaneous emission of nearby emitters. We also systematically evaluated various sample preparation steps to establish an optimized protocol to preserve cellular morphology and fluorescence signal. In summary, we propose a workflow for super-resolution imaging with mNeonGreen based on optimization of sample preparation, data acquisition and simple post-acquisition data processing. Application of our protocol enabled us to resolve the expected double band of bacterial cell division protein DivIVA, and to visualize that the chromosome organization protein ParB organized into sub-clusters instead of the typically observed diffraction-limited foci. We expect that our workflow allows a broad use of mNeonGreen for super-resolution microscopy, which is so far difficult to achieve.

EGFP oligomers as natural fluorescence and hydrodynamic standards.

EGFP oligomers are convenient standards for experiments on fluorescent protein-tagged biomolecules. In this study, we characterized their hydrodynamic and fluorescence properties. Diffusion coefficients D of EGFP1-4 were determined by analytical ultracentrifugation with fluorescence detection and by fluorescence correlation spectroscopy (FCS), yielding 83.4…48.2 µm(2)/s and 97.3…54.8 µm(2)/s from monomer to tetramer. A "barrels standing in a row" model agreed best with the sedimentation data. Oligomerization red-shifted EGFP emission spectra without any shift in absorption. Fluorescence anisotropy decreased, indicating homoFRET between the subunits. Fluorescence lifetime decreased only slightly (4%) indicating insignificant quenching by FRET to subunits in non-emitting states. FCS-measured D, particle number and molecular brightness depended on dark states and light-induced processes in distinct subunits, resulting in a dependence on illumination power different for monomers and oligomers. Since subunits may be in "on" (bright) or "off" (dark) states, FCS-determined apparent brightness is not proportional to that of the monomer. From its dependence on the number of subunits, the probability of the "on" state for a subunit was determined to be 96% at pH 8 and 77% at pH 6.38, i.e., protonation increases the dark state. These fluorescence properties of EGFP oligomeric standards can assist interpreting results from oligomerized EGFP fusion proteins of biological interest.

Scribed transparency microplates mounted on a modified standard microplate.

The immense cost effectiveness of using transparencies as analyte handling implements in microplate instrumentation offers the possibility of application even in resource-limited laboratories. In this work, a standard microplate was adapted to serve as the permanent base for disposable scribed transparencies. The approach is shown to ameliorate evaporation, which can affect assay accuracy when analytes need to be incubated for some time. It also offers assurance against fluorescence measurement errors due to the cross-talk of samples from adjacent wells.

Statistical optimization of green fluorescent protein production from Escherichia coli BL21(DE3).

An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.