**2. GFP-based biomarkers**

The term biomarker has accumulated a variety of definitions over the years. Herein, bio‐ markers are defined as genetically encoded molecular indicators of state that are linked to specific genes.The utility of GFP as a biomarker was first demonstrated using GFP reporter constructs [45]. When GFP is used as a transcription reporter, a cellular promoter drives ex‐ pression of the fluorescent protein resulting in fluorescent signal that temporally and locally reflects expression from the promoter *in vivo*. In the initial experiments, GFP cDNA [46] was expressed from the T7 promoter in *E. coli* or from the mec-7 (beta tubulin) promoter in *C. elegans* [45].*E. coli* cells fluoresced and the expression in *C. elegans* mirrored the pattern known from antibody detection of the native protein.Subsequently, GFP transcriptional re‐ porters have been used in a wide variety of organisms; GFP expression has minimal effect on the cells and can be monitored noninvasively using techniques such as fluorescence mi‐ croscopy and fluorescence assisted cell sorting (reviewed in [47]).

GFP fusion proteins (generated by combining the fluorescent protein coding region with the coding region of the cellular protein) are used as markers for visualization of intracellular protein tracking and interactions (reviewed in [47-49]).The GFP moiety may be N-terminal, C-terminal or even internal to the cellular protein.The availability of a color palette of fluo‐ rescent proteins allows multicolor imaging of distinct fluorescent protein fusions in the cel‐ lular environment. GFP fusion proteins are a major component of the molecular toolkit in cell biology.

#### **2.1. Using GFP as an** *in vivo* **solubility marker**

GFP has been used as a genetically encoded reporter for folding of expressed proteins.Ex‐ pression of recombinant proteins in *E.coli* is a powerful tool for obtaining large quantities of purified protein; however, some overexpressed recombinant proteins improperly fold and aggregate. Manipulation of conditions to generate soluble protein can be a laborious proc‐ ess. Directed evolution can be employed to increase the solubility of the recombinant pro‐ teins, but detection of specific mutants with improved solubility is a challenge.However, GFP biomarking can be utilized to address this challenge.Since GFP chromophore formation requires proper protein folding and GFP folds poorly when fused to misfolded proteins, flu‐ orescence of a GFP fusion protein can serve as an internal signal of a specific soluble (not aggregated) protein [26]. When used as a folding reporter, GFP is fused C-terminally to the protein of interest using a short linker between the two protein domains.Detection of fluo‐ rescence indicates that GFP domain is properly folded and that the protein of interest there‐ fore must be soluble.If the protein of interest misfolds and aggregates, the fused slowfolding GFP aggregates along with it and fluorescence is not detected. Therefore, this folding reporter assay can be used as a screening tool for soluble recombinant proteins in the context of directed evolution.

Some LOO-GFPs also show interesting reactions to ambient light. LOO11-GFP (beta strand 11 omitted) does not bind strand 11 when kept completely in the dark, but does bind it upon irradiation with light [43]. Raman spectroscopy showed that, in the dark, the chromophore assumes a *trans* conformation, and that light induces a switch to the native *cis* conformation. After irradiation, the chromophore relaxes back to the *trans* conformation. Following up on this result, [44] showed that using a circularly permuted LOO10-GFP construct (beta strand 10 omitted) and introducing two synthetic forms of strand 10, the wild-type strand and a strand with a mutation to cause yellow-shifted fluorescence, light irradiation increased the frequency of "peptide exchange" between the two strand 10 forms. The presence of this pep‐ tide exchange suggests that the cis/trans isomerization of the chromophore requires partial

The term biomarker has accumulated a variety of definitions over the years. Herein, bio‐ markers are defined as genetically encoded molecular indicators of state that are linked to specific genes.The utility of GFP as a biomarker was first demonstrated using GFP reporter constructs [45]. When GFP is used as a transcription reporter, a cellular promoter drives ex‐ pression of the fluorescent protein resulting in fluorescent signal that temporally and locally reflects expression from the promoter *in vivo*. In the initial experiments, GFP cDNA [46] was expressed from the T7 promoter in *E. coli* or from the mec-7 (beta tubulin) promoter in *C. elegans* [45].*E. coli* cells fluoresced and the expression in *C. elegans* mirrored the pattern known from antibody detection of the native protein.Subsequently, GFP transcriptional re‐ porters have been used in a wide variety of organisms; GFP expression has minimal effect on the cells and can be monitored noninvasively using techniques such as fluorescence mi‐

GFP fusion proteins (generated by combining the fluorescent protein coding region with the coding region of the cellular protein) are used as markers for visualization of intracellular protein tracking and interactions (reviewed in [47-49]).The GFP moiety may be N-terminal, C-terminal or even internal to the cellular protein.The availability of a color palette of fluo‐ rescent proteins allows multicolor imaging of distinct fluorescent protein fusions in the cel‐ lular environment. GFP fusion proteins are a major component of the molecular toolkit in

GFP has been used as a genetically encoded reporter for folding of expressed proteins.Ex‐ pression of recombinant proteins in *E.coli* is a powerful tool for obtaining large quantities of purified protein; however, some overexpressed recombinant proteins improperly fold and aggregate. Manipulation of conditions to generate soluble protein can be a laborious proc‐ ess. Directed evolution can be employed to increase the solubility of the recombinant pro‐ teins, but detection of specific mutants with improved solubility is a challenge.However,

croscopy and fluorescence assisted cell sorting (reviewed in [47]).

**2.1. Using GFP as an** *in vivo* **solubility marker**

unfolding of the protein.

cell biology.

**2. GFP-based biomarkers**

12 State of the Art in Biosensors - General Aspects

Split GFP may also be used to assay folding and solubility of a protein of interest in vivo by "tagging" the recombinant protein with the smaller portion of the split GFP sequence, and expressing the larger portion separately or adding it exogenously. The small size of a pro‐ tein tag makes it less likely to interfere with the folding and function of the protein of inter‐ est.In the split GFP complementation assay a large fragment of GFP folding reporter (GFP1-10 ) is coexpressed with tagged GFP protein (GFP11-protein x) [50]. As shown in Fig‐ ure 6, neither GFP1-10 nor the GFP11-tagged protein fluoresce alone; however, if both com‐ ponents are soluble,GFP1-10 and the GFP11-tagged protein reconstitute the native structure and fluorescence.For successful implementation of the assay, directed evolution of super‐ folder GFP1-10 was required. This resulted in GFP1-10 OPT which has an 80% increased sol‐ ubility over the corresponding superfolder GFP1-10.GFP OPT contains 7 new mutations (N39I, T105K, E111V, I128T, K166T, I167V and S205T) in addition to the superfolder muta‐ tions [50].Directed evolution of GFP11 resulted in GFP11 optima tag that had the dual prop‐ erties of 1) complementation with GFP1-10 OPT and 2) minimized perturbation of the protein of interest. Note that full-length GFP OPT was subsequently found to be more toler‐ ant of circular permutation and truncation than superfolder GFP [40].

**Figure 6.** Mechanism of LOO11-GFP (GFP1-10) as an in vivo solubility indicator for proteins tagged with strand 11 (GFP11). Modified with permission from [50].

In addition to providing a less laborious method for detecting protein variants and reaction conditions for generating soluble recombinant protein, the split GFP complementation assay also serves as an assay of aggregation in living cells. For example, aggregates of the microtu‐ bule associated protein tau are found in neurofibrillary tangles but their role in the patholo‐ gy of Alzheimer disease and Parkinson disease is not clear [51].The split GFP complementation assay enables monitoring of the aggregation process in living mammalian cells [52,53] and was validated using GFP1-10 and GFP11-tau variants.Cells containing soluble tagged protein show visible fluorescence but aggregates have little or no fluorescence. Pro‐ tein aggregates of GFP11-tau sequestered the GFP11 tag, leading to decreased complementa‐ tion of GFP1-10 and decreased fluorescence. Thus the split GFP complementation assay using tagged-GFP tau showed that it could be used as an *in vivo* model for studying factors that influence aggregation.

#### **2.2. GFP biomarkers for single molecule imaging**

It is also possible to utilize GFP biomarkers for single-molecule localization, a form of superresolution microscopy. High affinity single chain camelid antibodies (nanobodies) to GFP can be used to deliver organic fluorophores to GFP tagged proteins that are in turn used in single molecule "nanoscopy." [54, 55]. This novel approach combines the molecular specific‐ ity of genetic tagging with the high photon yield of the organic dyes. Additionally, by vary‐ ing the buffer conditions used, many organic dyes can become photoswitchable. The small size of camelid antibodies and their high affinity allow for access to regions that are general‐ ly inaccessible to conventional antibodies and targets that are expressed at very low levels [56].

One should caution that the overexpression of FRET biomarkers in transgenic animals car‐ ries some concerns that this could lead to the perturbation of endogenous signaling path‐ ways and even retardation of animal development [57]. Additionally, in compact tissue, such as the brain tissue, cell type identification is particularly tedious due the diffused ex‐ pression of the biomarkers.
