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Review

The HELP-UnaG Fusion Protein as a Bilirubin Biosensor: From Theory to Mature Technological Development

by
Paola Sist
1,
Ranieri Urbani
2,
Federica Tramer
1,
Antonella Bandiera
1 and
Sabina Passamonti
1,*
1
Department of Life Sciences, University of Trieste, 34127 Trieste, Italy
2
Department of Chemical and Pharmaceutical Sciences, University of Trieste, 34127 Trieste, Italy
*
Author to whom correspondence should be addressed.
Molecules 2025, 30(3), 439; https://doi.org/10.3390/molecules30030439 (registering DOI)
Submission received: 19 December 2024 / Revised: 9 January 2025 / Accepted: 10 January 2025 / Published: 21 January 2025
(This article belongs to the Special Issue Bioorganic Chemistry in Europe)
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Versions Notes

Abstract

:
HUG is the HELP-UnaG recombinant fusion protein featuring the typical functions of both HELP and UnaG. In HUG, the HELP domain is a thermoresponsive human elastin-like polypeptide. It forms a shield enwrapping the UnaG domain that emits bilirubin-dependent fluorescence. Here, we recapitulate the technological development of this bifunctional synthetic protein from the theoretical background of its distinct protein moieties to the detailed characterization of its macromolecular and functional properties. These pieces of knowledge are the foundations for HUG production and application in the fluorometric analysis of bilirubin and its congeners, biliverdin and bilirubin glucuronide. These bile pigments are metabolites that arise from the catabolism of heme, the prosthetic group of cytochromes, hemoglobin and several other intracellular enzymes engaged in electron transfer, oxygen transport and protection against oxygen free radicals. The HUG assay is a powerful, user-friendly and affordable analytical tool that alone supports research at each level of complexity or taxonomy of living entities, from enzymology, cell biology and pathophysiology to veterinary and clinical sciences.

Graphical Abstract

1. Introduction

HUG (acronym of HELP-UnaG) is a new recombinant fusion protein composed of two moieties, a human elastin-like polypeptide (HELP) carrier linked by a peptide bond to the N-terminal of UnaG [1]. HELP is an artificial polypeptide derived from the most regularly repeated motif of human elastin, resulting in an expression product with characteristic thermoresponsive properties [2]. UnaG is a natural protein first discovered in the muscle of the marine organism Japanese eel that binds the tetrapyrrolic compound bilirubin with high affinity emitting fluorescence [3]. HUG retains the functional properties of both parent proteins, though without identical parameters.
HUG was developed to address the need of a high-throughput, easy, affordable, sustainable and reliable analytical method to uniformly determine bilirubin and its congeners biliverdin and bilirubin glucuronide in living organisms and experimental models. This approach was aimed to avoid other methods that require the solvent-extraction of bilirubin and generate hazardous waste, like HPLC and LC-MS methods (e.g., Ref. [4]), and that require advanced laboratory equipment and a trained workforce. We addressed the need to avail of a simple, cheap and high-throughput assay, accessible to basic laboratories operating in pre-clinical or clinical research, requiring interference-free measurements. On these premises, we decided to employ UnaG for the fluorometric analysis of bilirubin, producing it as a recombinant fusion with the HELP carrier in bacteria and purifying it by exploiting the thermoresponsive properties of HELP.
Bilirubin is a yellow pigment found in animal plasma. Bilirubinemia is one of the most frequent blood tests undertaken to check the functional status of the liver [5], and values above 1 mg/dL are indicative of liver dysfunction and require further diagnostics [6]. Analyses are performed by an automated colorimetric method that measures the sum of bilirubin and its hepatic metabolite bilirubin glucuronide and, separately, bilirubin glucuronide, so that bilirubin concentrations are obtained by calculus. Remarkably, the latter fraction, also known as indirect bilirubin, is >90% under normal conditions. In the last decade, mild elevations of bilirubinemia that raise no disease concern have been found to be associated with a decreased disease risk [7], triggering strong interest in understanding the clinical relevance of this finding and in undertaking research to understand the factors that cause fine modulation of bilirubin metabolism and disposition. To this purpose, the use of pre-clinical experimental models is mandatory, but, in this case, the automated colorimetric method used in clinical chemistry cannot be applied to samples other than plasma. In several pre-clinical investigations, only HPLC-based methods could be used to isolate and quantify bilirubin and bilirubin glucuronide. Indeed, unlike the HPLC methods, the colorimetric diazo method is afflicted by interferences that undermine the quality of results in the normal range [8].
The aim of this review is to present the development of HUG from its conception to the implementation and application in different experimental models and research questions. HUG represents a case study of upward progress on the scale of technology readiness levels (TRL), a metric tool adopted by large R&D organizations or funding agencies to communicate the extent to which a given technology is scientifically validated and reliably applicable to the real world, i.e., outside the research laboratory [9,10]. There are nine TRL stages, spanning from the initial idea and outlining its theoretical background (TRL 1) to the description of the technology features and the experimental plan to create it (TRL 2). The first experimental demonstration and validation in a laboratory correspond to TRLs 3 and 4. The demonstration of the technology performance in a real environment(s) achieves TRL 5, and the creation of its pre-industrial prototype corresponds to TRL 6. TRLs 7 and 8 are associated with technology maturation at the industrial level (scaling-up, production process, large-scale technology applications, regulatory approval). TRL 9 is the stage of marketing. The wording and definitions are not rigid and can be adapted to specific science and technology domains, though the fundamental level distinctions are common, making the TRL scale a powerful tool to facilitate communication and management in innovation ecosystems [9].
Here, we present the technological development of HUG from its initial idea and its scientific background (TRL 1) to the current stage of a prototype (TRL 6) ready to be taken up and possibly further developed to full maturity (TRL 7–9) (Figure 1).

2. Basic Principles and Technology to Produce HELP-UnaG

The aim of creating a UnaG fusion product with the HELP carrier was based on our experience in producing recombinant HELP fusion products, such as HELPc [11], HELP Epidermal Growth Factor (HEGF) and HELP-RGD, which promoted cultured muscle cell differentiation [12]. One of the most attractive features of HELP fusion proteins is that they retain to some extent the thermoresponsive behavior known as the inverse thermal transition that can be exploited for their purifications.
The theoretical foundations required for the successful development of new recombinant HELP fusion proteins such as HUG are described below. They were not only the basis for the design, cloning, expression and purification of this new HELP fusion protein but were also the guide for further improvements and technological advancements. This description covers the first three levels of the TRL scale.

2.1. Engineered Elastin-like Polypeptides

Elastin is one of the main components of the extracellular matrix and one of the most studied structural proteins. It is characterized by rubbery elasticity and is a functional component of tissues such as blood vessels, skin and lungs [13]. Its precursor, tropoelastin, is rich in hydrophobic Val-Pro-Gly-Val-Gly (VPGVG) motifs and exhibits phase transition behavior at a low critical solution temperature (LCST). Tropoelastin is water-soluble below its transition temperature (Tt), whereas above it, the protein aggregates and separates as a second phase [14]. This peculiarity has inspired the study of engineered elastin-like polypeptides (ELPs), derived from the hydrophobic domain of bovine tropoelastin and retaining coacervation properties [15]. Like tropoelastin, ELP solutions also display LCST phase transition behavior [16,17,18]. This phenomenon, also known as the inverse thermal transition (ITT), is a phase transition of an ELP solution occurring at a specific solution temperature, Tt, which drives the formation of both intra- and intermolecular hydrophobic interactions, resulting in protein folding change and assembly [19]. Above the Tt, intermolecular polypeptide interactions are favored over polypeptide−solvent interactions, so that the protein separates from the solution. In this process, the regularly arranged water molecules of the hydrophobic hydration shell around ELPs become less ordered molecules, as in unperturbed bulk water [20], leading to a net increase in the entropy of the system.
ELPs are designed by a modular approach using variations of the VPGXG building block, where X can be any guest amino acid, except for proline, because of its chain conformation-disrupting properties [21], and the number and type(s) of repeated motifs can vary [22,23,24]. This paves the way for producing versatile ELP-based biomaterials [25], with fine-tuned hydrophobic indexes, thermosensitivity and gel-forming properties.
A sub-class of ELPs is given by engineered human elastin-like polypeptides (HELPs), featured by the hexapeptide repetitive domain Val-Ala-Pro-Gly-Val-Gly (VAPGVG) and, different from most of the other described ELPs, by the cross-linking domains found in human tropoelastin [26].

2.2. Design and Cloning of HELP in E. coli

HELP (MW = 44,886 Da) was designed to express two functional domains encoded by exons 23 and 24 of the human tropoelastin gene. The alanine-lysine-rich cross-linking domain, encoded by exon 23, is followed by the hexapeptide VAPGVG, encoded by exon 24 [26]. This bifunctional unit is repeated eight times [27] (Figure 2A). The sequence comprises a His-tag for exploiting affinity-based recognition by monoclonal antibodies. In addition, it was observed that the His-tagged HELP was expressed at higher levels with respect to the untagged HELP, resulting in an improved yield. The pEX8EL plasmid was used.
The synthetic HELP gene was cloned into a T5 promoter-based expression vector so that the inducible expression could be achieved by a lacIq strain (Figure 2B). The HELP expression construct was co-transformed into the NEBexpressIq E. coli strain. The conditions of the optimized HELP expression system are reported in Table 1.

2.3. Purification of HELPs

The scalability and standardization of the purification process determine the potential industrial production of elastin-like engineered proteins [28,29].
One of the possible methods for the purification of ELPs is the use of a His tag for immobilized metal affinity chromatography (IMAC) [30]. However, this method has some limitations and difficulties in scaling up, such as high costs due to the use of resins and the need for hazardous reagents (e.g., imidazole as a competitor molecule).
Non-chromatographic ELP purification methods like the organic extraction from whole cells and cell lysates with negligible contamination by nucleic acids or lipopolysaccharides are rare [31,32,33,34,35].
The preferred method for purifying ELPs takes advantage of their unique phase transition behavior, which achieves purification by inverse transition cycling (ITC) [36,37,38,39]. This simple and efficient method consists of hot centrifugation in a high ionic strength solution leading to a protein pellet made of ELP, followed by the pellet resuspension in cold water. The latter is centrifuged at low temperature to remove impurities. This hot–cold cycle can be repeated to obtain a pure product, though at the detriment of yield. Overall, the ITC method provides comparable or better results than the IMAC method, as reported by Ref. [40], showing that the ELP-conjugated superoxide dismutase (ELP-SOD) purified by ITC was equivalent or even better than that purified by Ni-NTA resin and ion exchange chromatography. With ITC, a scale-up from micrograms to milligrams is possible, and the technique can be optimized for high-throughput purification.

2.4. Physico–Chemical Features of HELP

Knowledge of the macromolecular parameters of HELP is the rational basis for establishing an efficient ITC-based purification protocol from bacterial extracts [14,26,41,42,43]. In addition, HELP is the reference polymer for the macromolecular characterization of new HELP fusion proteins and for the optimization of their purification and further technological utilization. We have described the aggregation properties and thermoresponsive behavior of the biopolymer HELP under different conditions using turbidimetry, differential scanning calorimetry, circular dichroism and dynamic light scattering [2,44,45].
The ability of HELP to undergo an inverse temperature transition under certain solution conditions, mainly at low and near-physiological salt concentrations, was determined by turbidimetric measurements. Under these conditions, HELP showed an inverse transition process very similar to that described for other elastin-like polypeptides [46,47]. Table 2 shows the Tt for HELP at different solvent conditions [45,48,49,50]. The HELP Tt was similar in different solvents, such as PBS, Tris and sodium phosphate buffer (NaPi), even with pH changes from 7.3 to 8.0. The presence of physiological salt concentrations (0.15 M NaCl) resulted in a marked effect on Tt and, remarkably, in an efficient coacervation process, which was evident in the form of a turbidity profile [2]. In contrast to other ELPs, where there are no cross-linking domains, the presence of a near-physiological NaCl concentration is essential to achieving or promoting the coacervation process of HELP and HELP-based fusions.
Differential scanning calorimetry (DSC) was used to characterize the inverse phase transitions of HELP in the presence of different NaCl concentrations [45]. A single endothermic peak with an asymmetric shape was recorded from which the Tt transition enthalpies (ΔHtr) and entropies (ΔStr) were determined (Table 3). In the entire salt concentration range (0.1–0.9 M NaCl), an entropy-driven process of dehydration was observed, as previously described for other elastin-like polypeptides [43,47]. Within this salt concentration range, HELP showed a constant linear increase in both transition enthalpy and entropy [2], as described by other authors for different ELPs [47], confirming that the presence of the cross-linking domains must be “neutralized” by at least one physiological salt concentration, making it an ELP. A very important result is that the low hydrophobicity of the HELP protein is associated with low values for the transition of energy and entropy, as generally observed for ELP polypeptides [50,51].
When different solvents were considered, i.e., Tris and NaPi as mixed solvents, no remarkable differences were found between the Tt values in the absence of NaCl salt (29–29.5 °C) (Table 3), which agrees well with the values obtained by turbidimetric techniques. The addition of the salt to the HELP solutions, responsible for shielding the charges and avoiding chain repulsions led to an increase in Tt (34–39 °C), with little differences between the different mixed solvents used.
Looking at the enthalpic and entropic results of the DSC (Table 3), it becomes clear that the effect of the ionic strength, destabilizing the water structure, results in less water available for the formation of cage-like structures around non-polar protein groups than in the lattice of unperturbed water. Thus, a remarkable entropy change was measured during the inverse transition process due to the loss of a relevant number of water molecules from the hydration structure, and a significant decrease in entropies was observed when NaCl salt was added to the solutions (Table 3).
Furthermore, significant differences were found between the enthalpies measured in different solvents, the values of which are related to the interactions between non-polar groups, ions and unperturbed water in the extended hydrophobic sphere and to the specific and different types of ordering in the local water (Table 3).
Circular dichroism (CD) spectra at different temperatures in aqueous and salt solutions were recorded [2,45]. The results showed the typical, well-documented CD profile of elastin and the short ELP polypeptides [16,52], which is characterized by a high percentage of random coil structure with a negative band around 200 nm (ππ* transition). The negative peak at 222 nm (nπ* transition) [16,53] was related to both α-helical segments (222 nm) and type I/type II β-turn and PP-II secondary structures at 225 nm. After the deconvolution of the CD spectra, the HELP sequence resembled the distribution of secondary structures predicted for human and bovine elastin, with a proportion of 20–29% α-helical domains, 60–63% of random coil and about 10% of β turn structures [48]. This pattern is typical of those of human and bovine elastin and similar to other short ELPs [2]. The secondary structure prediction for the HELP protein was calculated as the sum of the hydropathy values of all amino acids normalized by the number of amino acids in the sequence by the ProtParam (Expasy) program [54], which uses the GOR IV method to predict the secondary structure from the primary amino acid sequence [55]. The predicted results were consistent with the experimental data, resulting in an average value of 28% of α-helix content, about 70% of random coil and a low percentage of β structures, pointing to a prevalent disordered structure for the HELP protein.
Dynamic light scattering (DLS) was used to determine the hydrodynamic radius (RH) of HELP in a solution, as well as the dimensions of aggregates at different temperatures and concentrations. From the Stokes−Einstein theory, the diffusion coefficients D and then the hydrodynamic radius RH were calculated [56]. The percentage of the peak areas was obtained from intensity, volume and number distributions by nonlinear least-squares fitting.
Two modal size distributions were observed in the temperature range of 10−45 °C with an average RH value of 50 Å (78%) between 10 and 30 °C, which increases to about 1000 Å when the temperature rises to 45 °C due to the onset of the aggregation process [45]. If the temperature is raised above the Tt value, a complete aggregation process with a single modal distribution occurs, in which particles with an average RH value of more than 1000 Å are formed.

2.5. HELP Recombinant Fusion Proteins

Recombinant HELP fusion proteins retain the phase transition property of HELP. In this way, several recombinant HELP fusion proteins can be purified by the same ITC protocol, with minor modifications [37,57,58]. However, the feasibility of ITC-based purification must be assessed on a case-by-case basis to rule out any interference by the fused domain.
It was observed that all the HELP fusion proteins produced up to now were expressed in soluble form without the formation of inclusion bodies, as previously described for HELP [26]. Among the other advantages, the enhancement of protein expression was frequently observed as well as the retaining of the thermoresponsive behavior that allows for performing ITC-based purification. In addition, linkers and specific proteolytic sites can be inserted between HELP and the fusion domain for the release of this last (for example, see Ref. [49]).
Several bioactive ELPs as well as HELP fusion proteins have been developed for multiple purposes. The fused functional domain Arg-Gly-Asp (RGD) or the Epidermal Growth Factor (EGF) [12,59,60] have been exploited in in vitro cell cultures to enhance cell adhesion and differentiation [61,62,63,64]. Antimicrobial peptide fusions are used to develop bioengineered drugs [65,66].
On these premises, the design and production of a recombinant HELP-UnaG fusion was considered a feasible option. UnaG is produced by recombinant DNA technology as fusion products with histidine tails or proteins such as glutathione transferase [67], or maltose-binding proteins [68]. These adducts are ultimately enzymatically cleaved and removed by further chromatographic steps to obtain pure UnaG. We preferred to express a HELP-UnaG fusion product to obtain a new bi-functional polypeptide that may retain the properties of both carrier and fusion domains [1].

2.6. UnaG

UnaG is the first protein that can become fluorescent upon ligand binding obtained from a vertebrate. Its non-covalent binding to bilirubin, its physiological ligand, results in a green fluorescent complex. UnaG was first cloned and described in 2013, being isolated from the muscle fibers of the freshwater Japanese eel (Unagi, or Anguilla japonica) [3]. UnaG consists of a beta-barrel structure determined by 10 antiparallel beta-strands. In holoUnaG, the bilirubin is in the center of the cavity formed by the beta-barrel. As can be seen from the atomic structure of holoUnaG, the high binding affinity between bilirubin and UnaG is determined by numerous interactions based on the formation of hydrogen bonds with appropriately and orderly positioned water molecules and amino acid residues [3].
It belongs to the family of fatty acid-binding proteins, binds bilirubin with high affinity and specificity and produces fluorescence even under anoxic conditions [69].
Since its discovery, UnaG has attracted the attention of the scientific world due to its potential for clinical and technological applications in the preclinical field.
The high affinity (Kd = 98 pM) of the protein to its ligand makes it an excellent tool for direct dosing of unconjugated bilirubin even in blood samples. The fluorescence emission is not affected by the presence of other molecules such as hemoglobin, and thus sample processing takes less time. In addition, the UnaG binding with bilirubin is much stronger than the binding of bilirubin with albumin, so no deproteinization of the plasma is required either [3].
The expression of UnaG has been performed and optimized both in bacteria, to employ the purified apoprotein for clinical applications, and in eukaryotic cells, for technological applications, to study cellular heme metabolism or as a reporter (Table 4). In bacteria, the apo-protein (MW = 15,581 Da) is expressed with a tag that allows for affinity purification. The different uses and related purification methods are presented in Table 4.

3. HUG Assay: Features and Use for the Fluorometric Analysis of Bile Pigments

We tested and validated all parameters of the HUG and of the assay based on this tool in five distinct projects, culminating with the publication of research and methodology articles. First, we performed the physico–chemical characterization of HUG for both its general macromolecular properties and as a specific bilirubin-dependent fluorophore [45]. Then, we standardized its lab-scale production [124]. We established a procedure for preparing quality-controlled bilirubin standard solutions spanning the range of 10−3–10−9 M [125]. We set up a method for the nanoscale analysis of bilirubin by HUG that yields results matching the standard bilirubin analysis in clinical chemistry [126], and we upgraded it for the combined analysis of both biliverdin and bilirubin in the same sample [127]. In another project, we included the validated analysis of bilirubin glucuronide by upgrading the HUG assay with the enzyme β-glucuronidase [128].

3.1. Design and Cloning of HUG in E. coli

The engineered HELP protein was fused with the UnaG eel protein to obtain a chimeric polypeptide, which was named HUG [1]. For the realization of HUG, the recombinant gene of the HELP polypeptide was fused with the 139 amino acid-coding sequence of the UnaG bilirubin-binding protein (accession number BAN57322.1; GenBank), exploiting the unique DraIII site in the expression vector that allows for the in-frame insertion of the polypeptide at the C terminus (Figure 3).
The fusion product was expressed in the C3037 E. coli strain, and the conditions of the optimized HUG expression system are the same as those reported in Table 1.

3.2. The Protocol to Prepare Standardized Lots of HUG

The HUG recombinant protein was produced and purified as reported [124]. The protocol for the purification of HUG from induced E. coli biomass extracts is based on the addition of NaCl to the soluble fraction of the extract to lower the inverse transition temperature and promote the coacervation process. Large HUG aggregates are sedimented by centrifugation at low speed (<10,000 rpm). The resulting pellet is redissolved by cooling. This temperature-dependent transition from the liquid to solid phases is repeated several times to obtain the purified HUG protein (Figure 4). The purification protocol differs slightly from that intended for HELP since it requires the use of sodium deoxytaurocholate to remove lipid residues that the fatty acid-binding protein UnaG may have co-precipitated.
The final product is subjected to quality control to evaluate purity, concentration and bilirubin-dependent-specific fluorescence. Under these standardized conditions, the HUG exhibits a specific UV–Vis extinction coefficient ε280 = 18.747 and a bilirubin-dependent-specific fluorescence of 11.663 A.U./μg. The protein yield averages 200 mg per liter of bacterial culture. The production of 0.5 g HUG requires 5 days of experimental work when the bacterial clone is already available [124]. Considering that the standard protocol for nanoscale fluorometric analysis of bilirubin requires 1 mg of HUG for a 96-well plate suitable for the analysis of 24 samples (among which the calibration standards), each in quadruplicate, a lot of 1 g of HUG obtained from 5 L of bacterial culture enables the analysis of 24,000 samples.

3.3. Physico–Chemical Features of HUG

The macromolecular characterization of HUG [45] has provided insights into the solution properties of the protein chain and into its capacity to form a complex with bilirubin. First, as in the case of the HELP polypeptide, thermodynamic and spectroscopic techniques were used to analyze the reverse-transition process of the HUG. In addition, the binding capacity of the HUG was investigated under various solution conditions using fluorometric methods.
The features of the HUG macromolecule were predicted using the program ProtParam (Expasy) based on the primary structure of the protein. With respect to the HELP biopolymer, the secondary structure distribution for HUG showed a greater proportion of β-strand conformation (10%) due to the UnaG contribution. These calculations showed, in addition, that both sequences of HELP and UnaG retain their secondary structure distribution in the HUG fusion protein.
The CD spectra of the HUG solutions showed the typical shape previously observed for HELP biopolymers [2] and generally shared by the ELP polypeptides [16,53]. Less pronounced negative bands at 200 nm and 222 nm for HUG compared to HELP were recorded due to the changes in secondary structure distributions with a lower proportion of α-helix and random coil sequences in the HUG polypeptide [45]. Accordingly, molecular modeling performed with AlfaFold2 in vacuo [129] returned the tertiary structure, as shown in Figure 5.
The inverse thermal transition of HUG was investigated using turbidimetry and DSC techniques. As shown for HELP, the HUG biopolymer underwent the typical hydrophobic process as a function of temperature (coacervation) with a strong increase in turbidity. Under different solvent conditions (PBS and Tris/NaCl), HUG showed little change in Tt compared to HELP solutions, with a value in the range of 31.9–33 °C [45].
The entropic and enthalpic contributions were determined from the DSC measurement. The lower chain hydrophobicity calculated for HUG compared to HELP resulted in a significant decrease in ΔHtr of HUG (in PBS, pH = 7.4), mainly due to the different charged groups present, while the similar number of water molecules in the solvation spheres made a similar contribution to ΔStr [45].
Using the DLS technique, the dimensions of the HUG aggregates of different sizes at different temperatures and concentrations were measured. With an average particle radius of 62, 250 and >150 Å, a three-modal size distribution was observed as a function of temperature [45].
The fluorescence spectral characteristic of the complexes of bilirubin with UnaG and HUG is the same, as indicated by the peak values derived from their excitation and emission spectra, with excitation maxima at 498 nm for both UnaG and HUG and 527 or 530 nm for UnaG and HUG, respectively [3], [1]. A refined estimate of the binding of UnaG with bilirubin revealed that the Kd of the complex is 0.031–0.098 nM [3,67] which results from the parameter of a simple hyperbola. Although the Kd value of the HUG–BR complex (1.1–1.7 nM) [1,45] is two-to-three orders of magnitude higher than that of UnaG-BR, it is still lower than that of the albumin–bilirubin complex (45 nM) [130]. Therefore, the HUG can efficiently displace Br from BSA and enable the determination of bilirubin even in biofluids in which bilirubin forms a complex with serum albumin (typically in blood).

3.4. The Protocol to Prepare Standard Solutions of Bilirubin

Methods that analyze bilirubin aqueous solutions in the nanomolar scale encounter the challenge of calibration, which impacts the assay performance, reliability and comparison of results across different methods and real samples. Bilirubin is a lipophilic compound with an upper limit of solubility in water <100 nM and a marked instability. Bilirubin standards in the nanomolar range are not commercially available. Moreover, we found quite scant published information on how bilirubin standards are prepared in different methods. We addressed this gap of knowledge by establishing a standardized protocol for the preparation of quality-controlled standard bilirubin solutions [125]. To overcome chemical instability, we supplemented standards with low amounts of bovine serum albumin, which does not interfere with the assay because the affinity HUG for bilirubin is higher than for albumin(s), and therefore any albumin-bound bilirubin molecule will be transferred to HUG at equilibrium.

3.5. The HUG-Based Method for the Nanoscale Analysis of Bilirubin

The HUG method for the nanoscale analysis of bilirubin [126] exploits the 96-well plate configuration, as described in Figure 6. A HUG solution is distributed in the plate’s wells. Standard solutions of bilirubin are added at concentrations 0–50 nM to obtain a calibration curve. The samples are distributed in the wells and the emitted fluorescence is quantified by a microplate reader.
The validation parameters of the method are shown in Table 5.
After extracting information from a recent review that compares 42 methods for the analysis of bilirubin [131], in terms of LOQ, the HUG-based method ranks third among 27 fluorescence methods and is comparable to the best HPLC method coupled to MS, but it is outperformed by three electrochemical methods based on nanomaterials. However, a crucial feature of the HUG assay is that it does not require sample preparation, advanced equipment or specialized technical skills.
The robustness of the method was assessed with five common buffered solutions. The angular coefficients (nM−1) of the calibration curves were 551–663. The supplementation of phosphate-buffered saline solution (PBS) with bovine serum albumin led to an increased angular coefficient (785 nM−1), whereas the addition of the surfactant Tween 20 at 1% produced an angular coefficient of 7.5 nM−1, likely due to incompatibility with HUG. Other surfactants, such as 1% Triton X-100 or 0.2 mM sodium taurocholate, had no major impact on this parameter. Quite remarkably, DMSO up to 30% was tolerated, which is a major advantage when assessing bilirubin in the presence of other lipophilic compounds.
In another study, we demonstrated further details about the robustness and specificity of the method, showing that a series of drug molecules used to inhibit the hepatic uptake of bilirubin did not interfere with the assay, i.e., estradiol 17-β-glucuronide, pravastatin, ketoprofen, cyanidin 3-glucoside, indomethacin and taurocholate [128].
When applied to human plasma, the precision of this method (Table 5) enables us to discriminate the bilirubin phenotype pattern linked to genetic polymorphisms of the gene UGT1A1, which determines graded mean increases of conjugated bilirubin in the blood. Similarly, the method enables us to detect well-known sex-related differences in bilirubinemia [126].

3.6. The Upgraded HUG Method for the Analysis of Biliverdin and Bilirubin Glucuronide

The configuration of the HUG assay in 96-well plates enables us to prepare wells not only with HUG but also with HUG plus biliverdin reductase and NADPH to obtain the total conversion of biliverdin to bilirubin [127]. Similarly, wells can be prepared with HUG and β-glucuronidase that catalyze the hydrolysis of bilirubin glucuronide(s) to bilirubin [128]. The presence of HUG in the reaction wells traps the main reaction product bilirubin and therefore drives both reactions to completion. Appropriate controls must be included to pinpoint unwanted fluorescence artifacts.

3.7. The Technological Readiness Level of the HUG Assay

The scheme presented in Figure 7 provides an overview of the experimental steps undertaken to characterize the performance of the HUG assay, framing them in the technology readiness levels paradigm.

4. Bile Pigment Analysis in Biology and Medicine: Theory and Applications

The HUG assay opens the possibility for any laboratory to use the same method for advanced bile pigment analysis spanning a wide arch of biomedical translational research and reaching clinical studies (Figure 8).
This is a great advantage for a research group since a given hypothesis can be tested in experimental models of different degrees of complexity (e.g., isolated proteins or cells; micro-physiological systems like organoids, experimental animals and so on) by availing of the same analytical approach. As a result, not only can the reproducibility of the results be repeatedly tested and confirmed but even more new biological principles may emerge. Indeed, we anticipate the improvement of our still imprecise understanding of the fine regulation of the production of bile pigments from heme catabolism and hepatic bilirubin elimination. The biology of these processes is summarized below.

4.1. Sources of Heme

Heme plays vital roles in aerobic life, such as the binding of oxygen for cellular storage (myoglobin), scavenging reactive oxygen species (catalase, peroxidase), electron transport (cytochromes), synthesis of bioactive molecules (cyclooxygenase, nitric oxide synthase), regulation of gene expression (heme response elements) and transport of oxygen to the tissues via blood circulation (hemoglobin). Many cellular functions are influenced by the rate of heme catabolism, such as cellular oxidative stress response, inflammation and related diseases of the central and peripheral nervous system, cardiovascular and respiratory systems and cancer [133]. Since bilirubin is eliminated by biliary excretion after hepatic metabolism, bilirubin is a primary biomarker of liver disease. The largest pool of heme (about 75%) is found in hemoglobin and is released in erythrocyte turnover in the spleen. Accordingly, hemolytic diseases produce large amounts of bilirubin. The remaining fraction of heme derives from the ubiquitous cellular catabolism of heme-bound enzymes [134], a process that is regulated by a multitude of stress factors.

4.2. Synthesis of Bile Pigments

Both biliverdin and bilirubin are tetrapyrrolic molecules synthesized in animal cells from heme in a two-step reaction pathway, catalyzed by heme oxygenase (HO) (EC: 1.14.14.18) and biliverdin reductase (E.C. 1.3.1.24) (Figure 9). The products of HO are biliverdin, carbon monoxide and ferric ion in equimolar stoichiometry. The diffusion of CO in the body and the atmosphere drives the HO reaction to completion. In mammals, the reaction of biliverdin reductase is essentially irreversible, with a complete conversion of hydrophilic biliverdin to lipophilic bilirubin [135]. This may be ascribed to the fact that bilirubin diffuses from the cells into the blood, where it forms a high-affinity complex with serum albumin (45 nM) [130], leaving the unbound fraction to nM concentrations [136].
HO is expressed in non-erythroid tissues in two isoforms, HO-1 and HO-2, encoded by two genes [135,137], which catalyze the same reaction though with different enzyme kinetics parameters. The former is inducible under a variety of cellular stress conditions, whereas HO-2 is seen as the isoform that covers the basal needs of heme turnover [137]. HO-1 induction plays a major role in cytoprotection, observed at all levels of complexity from in vitro experimental models to the beneficial effects of all major apparatus of the human organism [138]. An excessive induction of HO-1 can, however, be detrimental, mostly because of the excessive production of free iron [139]. Indeed, some anticancer drugs do act by abnormally increasing the expression and activity of HO, leading to desired cell death. By using HUG for the combined analysis of biliverdin and bilirubin, we could demonstrate that the upregulation of HO mRNA and proteins was paralleled with a significant increase of intracellular biliverdin in the metastatic cancer cell line [139]. There is a huge research interest in understanding the regulation of HO and the long-range impact of changes in its expression, as indicated by >16,000 items in PubMed. Adding the analysis of biliverdin in cells, tissues and biofluids to the current practice of quantifying HO mRNA or proteins provides a deeper phenotypic understanding of this metabolic hot point.

4.3. Bilirubin Elimination

Bilirubin is selectively taken up into the liver by a mechanism that is still poorly characterized, with questionable involvement of sinusoidal membrane transporters known as organic anion transporting polypeptide 1B1 (OATP1B1, SLCO1B1) and OATP1B3 (SLCO1B3) [140]. The likely involvement of other membrane transporters has been demonstrated by analyzing both bilirubin and bilirubin glucuronide in the outflow of the isolated perfused rat liver by a HUG assay [128]. In the liver, bilirubin is metabolized to bilirubin mono- and di-glucuronide by the specific UDP-glucuronosyltransferase UGT1A1 (EC 2.4.1.17) and excreted into the bile by the biliary ATP-dependent transporter MRP2 (ABCC2) [134,140].

4.4. The Bioactivity of Bile Pigments

4.4.1. Biliverdin

Biliverdin is the direct conversion product of heme catalyzed by a family of enzymes known as heme oxygenase. In the cytoplasm of the cell, biliverdin is normally converted to bilirubin by biliverdin reductase and subsequently excreted into the plasma or extracellular environment.
Biliverdin is a more hydrophilic compound than bilirubin and is non-toxic; moreover, it can be excreted as such in both bile and urine [141]. Together with bilirubin, it is known for its antioxidant, anti-inflammatory and anti-apoptotic effects.
The antioxidant action of biliverdin may protect the brain from ischemia-reperfusion injury via the Nrf2/A20/eEF1A2 axis and inhibition of pyroptosis [142]. Antioxidant activity was also detected in the erythrocytes of 9-month-old Bvra-/- mice, in which oxidative stress was significantly reduced [143]. In addition, the antioxidant activity was associated with the BV/BR cycle in cells, and thus with the activity of enzymes involved in this process, such as heme oxygenase and biliverdin reductase, which can reduce the apoptotic activity of cisplatin in NRK-52E renal cells [144]. It has also been shown that this molecule can act as an antitumor molecule via a mechanism related to its antioxidant activity by modulating the angiogenic pathway [144]. The anti-inflammatory effect of BV is due to its ability to induce and bind BVR present on the macrophage membrane, which in turn activates the PI3K–Akt signaling pathway that triggers the anti-inflammatory response [145].

4.4.2. Bilirubin: Observational Studies

In the absence of hemolytic anemias or liver diseases that cause different patterns of hyperbilirubinemia and jaundice in adult life, mild elevations of total bilirubinemia within the physiological range are associated with the decreased risk of several disease conditions and different populations. The first observation of a negative correlation between cardiovascular diseases and total bilirubinemia was reported in 1994 [146]. Several other clinical studies followed that investigated correlations with a wider disease spectrum. A series of metanalyses established that the participant groups with the highest bilirubin levels had a significant disease risk. For example, data from 12 prospective studies involving 368,567 participants identified a decreased cardiovascular disease risk (by 25%) [147]; data from 11 cohort studies involving 263,596 participants identified a decreased stroke risk (by 15%) [148]; and data from 10 studies involving 79,508 individuals found a decreased risk of metabolic syndrome (by 30%) and type 2 diabetes (by 22%) [149].
Of related interest is also the observation of a significant negative correlation between total bilirubin and the systemic immune–inflammation index that reflects the balance between inflammation and immune response [150].
When available, for instance in Refs. [147,150,151], the dose–response curve showed the strongest decrease of disease risk occurred in the hypobilirubinemic range, i.e., <10 µM, taken as the reference normal value. Given that subtle changes of bilirubinemia at values <10 µM are related to sizeable changes in the disease risk [7,152], it is evident that accurate measurements of total bilirubinemia and its fractions are necessary for correct understanding of study results.
Similar beneficial effects are observed in subjects presenting Gilbert’s syndrome. This is defined as primary mild hyperbilirubinemia, i.e., the absence of hemolytic and/or liver diseases. It is an autosomal recessive hereditary condition found in about 7% of the European population and is asymptomatic. The molecular defect consists of mutations in the gene-encoding uridine diphosphate glycosyltransferase 1 (UGT1A1) [153,154]. Subjects carrying this genetic signature are shown to have improved endothelial function [155], fewer cardiovascular disease events [156], improved serum lipid profile [157], presumably enhanced lipid catabolism rate [158], lower inflammatory biomarkers [159] and lower risk of death [160].

4.4.3. Bilirubin: Mechanistic Studies

Bilirubin is an antioxidant both in itself [161,162,163,164] and acting indirectly as it reduces the production of reactive oxygen species by the ubiquitous membrane enzyme NADPH oxidase (E.C. 1.6.3.1) [165,166]. Bilirubin acts as an electrophile binding to protein thiol groups, thus regulating the cellular redox state [167], and as a scavenger of reactive oxygen species, as demonstrated in in vivo mice [84]. It also has antibacterial activity against pathogenic bacteria of the gut microbiota [168]. Molecular studies indeed identified bilirubin as a ligand of several nuclear and cytoplasmic receptors, such as the aryl hydrocarbon receptor (AhR), the peroxisome proliferator-activated receptor-alpha (PPARα), the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) and some others, as reviewed in Ref. [169]. These multiple functions result in fine regulation of metabolic genes related to lipid catabolism and glucose metabolism, especially in the liver [170].
The full spectrum of the bilirubin molecular targets remains to be explored.

5. Perspectives

At this stage of technological maturity, we can consider that the HUG-based assay for the analysis of bilirubin, bilirubin glucuronide and biliverdin can be regarded as an affordable emerging technology [171] that can not only enhance the analytical arsenal of laboratories active in life sciences but also add value to clinical diagnostics when an accurate quantification of each bile pigment is necessary and HPLC methods are the ultimate resource, as seen in Ref. [4].
Some limitations need more time and effort to be resolved. At present, the HUG biopolymer is not commercially available. This may limit its wide use and slow down its progress to TRL 7 and beyond. Though it can be easily produced by a standardized procedure at a low cost, its wide use is limited; thus, it must progress to TRL 7 and beyond. A corollary to this not-insurmountable limitation is that the HUG assay is so far intended to be operated only in laboratory settings in the pre-clinical or clinical research domains. At present, the HUG assay serves as a companion diagnostic device for precision medicine, given its accuracy and specificity in detecting three bile pigments. A larger number of assay demonstrations in addressing the significance of bile pigments as biomarkers of disease risk is needed to consolidate its position at TRL 6 and therefore move to industrial production (TRL 7), starting the procedures for regulatory approval (TRL 8).
Further cycles of technology innovation by advanced engineering approaches are needed to integrate HUG into microfluidic devices for use as a biomarker sensor in in vitro micro-physiological systems (e.g., organ-on-chip) or in portable, non-invasive analytical devices to measure bilirubin [172,173].
Improvements attainable in the short–medium term are possible due to the versatile properties of the HELP domain that enable the formation of composite materials [1,174,175], its shaping to 3D configuration by cross-linking [176] and its modulable thermoresponsive properties [48]. For example, it is possible to prototype HUG-coated multi-well plates or microfluidic systems to engineer composite biomaterials and develop multi-sensor devices by fusing the HELP scaffold with other on-demand functional domains [177].
At its present stage of technological readiness, the HUG assay is a reliable and invaluable resource for screening clinical biobanks of serum and other biofluids, as well as of other biological materials (e.g., experimental and livestock animals; samples of toxicological in vitro and in vivo tests), providing an immediate opportunity to step toward TRL 7 and beyond.

Author Contributions

Conceptualization, S.P.; writing—original draft preparation, S.P., P.S., R.U. and F.T.; writing—review and editing, S.P., P.S., R.U., F.T. and A.B.; visualization, P.S.; supervision, S.P.; project administration, S.P.; funding acquisition, S.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the European Union via NextGenerationEU (iNEST—Interconnected Nord-Est Innovation Ecosystem, ID ECS0000004-CUP J43C22000320006). The views and opinions expressed are, however, those of the authors only and do not necessarily reflect those of the European Union or the European Research Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Bandiera, A.; Corich, L.; Tommasi, S.; De Bortoli, M.; Pelizzo, P.; Stebel, M.; Paladin, D.; Passamonti, S. Human Elastin-like Polypeptides as a Versatile Platform for Exploitation of Ultrasensitive Bilirubin Detection by UnaG. Biotechnol. Bioeng. 2020, 117, 354–361. [Google Scholar] [CrossRef] [PubMed]
  2. Bandiera, A.; Sist, P.; Urbani, R. Comparison of Thermal Behavior of Two Recombinantly Expressed Human Elastin-like Polypeptides for Cell Culture Applications. Biomacromolecules 2010, 11, 3256–3265. [Google Scholar] [CrossRef]
  3. Kumagai, A.; Ando, R.; Miyatake, H.; Greimel, P.; Kobayashi, T.; Hirabayashi, Y.; Shimogori, T.; Miyawaki, A. A Bilirubin-Inducible Fluorescent Protein from Eel Muscle. Cell 2013, 153, 1602–1611. [Google Scholar] [CrossRef] [PubMed]
  4. Schoissengeier, V.; Maqboul, L.; Weber, D.; Grune, T.; Bürkle, A.; Moreno-Villaneuva, M.; Franceschi, C.; Capri, M.; Bernhard, J.; Toussaint, O.; et al. Association between Bilirubin and Biomarkers of Metabolic Health and Oxidative Stress in the MARK-AGE Cohort. iScience 2024, 27, 110234. [Google Scholar] [CrossRef] [PubMed]
  5. Burra, P.M.A. Dynamic Tests to Study Liver Function. Eur. Rev. Med. Pharmacol. Sci. 2004, 8, 19–21. [Google Scholar] [PubMed]
  6. Fevery, J. Bilirubin in Clinical Practice: A Review. Liver Int. 2008, 28, 592–605. [Google Scholar] [CrossRef]
  7. Vítek, L. Bilirubin as a Predictor of Diseases of Civilization. Is It Time to Establish Decision Limits for Serum Bilirubin Concentrations? Arch. Biochem. Biophys. 2019, 672, 108062. [Google Scholar] [CrossRef] [PubMed]
  8. Levitt, D.G.; Levitt, M.D. Development of a Pharmacokinetic Model That Accounts for the Plasma Concentrations of Conjugated and Unconjugated Bilirubin Observed in a Variety of Disease States. Clin. Exp. Gastroenterol. 2023, 16, 277–289. [Google Scholar] [CrossRef] [PubMed]
  9. Héder, M. From NASA to EU: The Evolution of the TRL Scale in Public. Sector Innovation. Innov. J. 2017, 22, 1–23. [Google Scholar]
  10. Savoini, A.; Tronchin, A. Definition of technology readiness levels (trl) and application to biomedical field. In Trans2care. Cross-Border Italy-Slovenia Biomedical Research. Are We Ready for Horizon 2020? Gustincich, S., Lah Turnšek, T., Passamonti, S., Peterlin, B., Pišot, R., Storici, P., Eds.; EUT Edizioni Università di Trieste: Trieste, Italy, 2014; pp. 421–424. Available online: http://hdl.handle.net/10077/10261 (accessed on 9 January 2025).
  11. D’Andrea, P.; Scaini, D.; Severino, L.U.; Borelli, V.; Passamonti, S.; Lorenzon, P.; Bandiera, A. In Vitro Myogenesis Induced by Human Recombinant Elastin-like Proteins. Biomaterials 2015, 67, 240–253. [Google Scholar] [CrossRef]
  12. D’Andrea, P.; Sciancalepore, M.; Veltruska, K.; Lorenzon, P.; Bandiera, A. Epidermal Growth Factor—Based Adhesion Substrates Elicit Myoblast Scattering, Proliferation, Differentiation and Promote Satellite Cell Myogenic Activation. Biochim. Biophys. Acta Mol. Cell Res. 2019, 1866, 504–517. [Google Scholar] [CrossRef]
  13. Depenveiller, C.; Baud, S.; Belloy, N.; Bochicchio, B.; Dandurand, J.; Dauchez, M.; Pepe, A.; Pomès, R.; Samouillan, V.; Debelle, L. Structural and Physical Basis for the Elasticity of Elastin. Q. Rev. Biophys. 2024, 57, e3. [Google Scholar] [CrossRef] [PubMed]
  14. Ozsvar, J.; Yang, C.; Cain, S.A.; Baldock, C.; Tarakanova, A.; Weiss, A.S. Tropoelastin and Elastin Assembly. Front. Bioeng. Biotechnol. 2021, 9, 643110. [Google Scholar] [CrossRef] [PubMed]
  15. Urry, D.W. Elastic Molecular Machines in Metabolism and Soft-Tissue Restoration. Trends Biotechnol. 1999, 17, 249–257. [Google Scholar] [CrossRef] [PubMed]
  16. Urry, D.W.; Long, M.M.; Cox, B.A.; Ohnishi, T.; Mitchell, L.W.; Jacobs, M. The Synthetic Polypentapeptide of Elastin Coacervates and Forms Filamentous Aggregates. Biochim. Biophys. Acta (BBA) Protein Struct. 1974, 371, 597–602. [Google Scholar] [CrossRef]
  17. Urry, D.W. Free Energy Transduction in Polypeptides and Proteins Based on Inverse Temperature Transitions. Prog. Biophys. Mol. Biol. 1992, 57, 23–57. [Google Scholar] [CrossRef] [PubMed]
  18. Roberts, S.; Dzuricky, M.; Chilkoti, A. Elastin-like Polypeptides as Models of Intrinsically Disordered Proteins. FEBS Lett. 2015, 589, 2477–2486. [Google Scholar] [CrossRef] [PubMed]
  19. Li, B.; Alonso, D.O.V.; Daggett, V. The Molecular Basis for the Inverse Temperature Transition of Elastin11Edited by A. R. Fersht. J. Mol. Biol. 2001, 305, 581–592. [Google Scholar] [CrossRef] [PubMed]
  20. Perticaroli, S.; Ehlers, G.; Jalarvo, N.; Katsaras, J.; Nickels, J.D. Elasticity and Inverse Temperature Transition in Elastin. J. Phys. Chem. Lett. 2015, 6, 4018–4025. [Google Scholar] [CrossRef] [PubMed]
  21. Garanger, E.; Lecommandoux, S. Emerging Opportunities in Bioconjugates of Elastin-like Polypeptides with Synthetic or Natural Polymers. Adv. Drug Deliv. Rev. 2022, 191, 114589. [Google Scholar] [CrossRef]
  22. Meyer, D.E.; Chilkoti, A. Quantification of the Effects of Chain Length and Concentration on the Thermal Behavior of Elastin-like Polypeptides. Biomacromolecules 2004, 5, 846–851. [Google Scholar] [CrossRef] [PubMed]
  23. Park, J.E.; Won, J.I. Thermal Behaviors of Elastin-like Polypeptides (ELPs) According to Their Physical Properties and Environmental Conditions. Biotechnol. Bioprocess Eng. 2009, 14, 662–667. [Google Scholar] [CrossRef]
  24. López Barreiro, D.; Folch-Fortuny, A.; Muntz, I.; Thies, J.C.; Sagt, C.M.J.; Koenderink, G.H. Sequence Control of the Self-Assembly of Elastin-like Polypeptides into Hydrogels with Bespoke Viscoelastic and Structural Properties. Biomacromolecules 2023, 24, 489–501. [Google Scholar] [CrossRef] [PubMed]
  25. Kowalczyk, T.; Hnatuszko-Konka, K.; Gerszberg, A.; Kononowicz, A.K. Elastin-like Polypeptides as a Promising Family of Genetically-Engineered Protein Based Polymers. World J. Microbiol. Biotechnol. 2014, 30, 2141–2152. [Google Scholar] [CrossRef]
  26. Bandiera, A.; Taglienti, A.; Micali, F.; Pani, B.; Tamaro, M.; Crescenzi, V.; Manzini, G. Expression and Characterization of Human-elastin-repeat-based Temperature-responsive Protein Polymers for Biotechnological Purposes. Biotechnol. Appl. Biochem. 2005, 42, 247–256. [Google Scholar] [CrossRef] [PubMed]
  27. Bandiera, A. Assembly and optimization of expression of synthetic genes derived from the human elastin repeated motif. Prep. Biochem. Biotechnol. 2010, 40, 198–212. [Google Scholar] [CrossRef] [PubMed]
  28. Mahmoodi, S.; Pourhassan-Moghaddam, M.; Wood, D.W.; Majdi, H.; Zarghami, N. Current Affinity Approaches for Purification of Recombinant Proteins. Cogent Biol. 2019, 5, 1665406. [Google Scholar] [CrossRef]
  29. Haas, S.; Desombre, M.; Kirschhöfer, F.; Huber, M.C.; Schiller, S.M.; Hubbuch, J. Purification of a Hydrophobic Elastin-like Protein Toward Scale-Suitable Production of Biomaterials. Front. Bioeng. Biotechnol. 2022, 10, 878838. [Google Scholar] [CrossRef] [PubMed]
  30. Charlton, A.; Zachariou, M. Immobilized Metal Ion Affinity Chromatography of Native Proteins. In Affinity Chromatography; Humana Press: Totowa, NJ, USA, 2008; pp. 25–36. [Google Scholar]
  31. Verheul, R.; Sweet, C.; Thompson, D.H. Rapid and Simple Purification of Elastin-like Polypeptides Directly from Whole Cells and Cell Lysates by Organic Solvent Extraction. Biomater. Sci. 2018, 6, 863–876. [Google Scholar] [CrossRef] [PubMed]
  32. Sweet, C.; Aayush, A.; Readnour, L.; Solomon, K.V.; Thompson, D.H. Development of a Fast Organic Extraction–Precipitation Method for Improved Purification of Elastin-like Polypeptides That Is Independent of Sequence and Molecular Weight. Biomacromolecules 2021, 22, 1990–1998. [Google Scholar] [CrossRef]
  33. Riziotis, I.G.; Lamprou, P.; Papachristou, E.; Mantsou, A.; Karolidis, G.; Papi, R.; Choli-Papadopoulou, T. De Novo Synthesis of Elastin-like Polypeptides (ELPs): An Applied Overview on the Current Experimental Techniques. ACS Biomater. Sci. Eng. 2021, 7, 5064–5077. [Google Scholar] [CrossRef]
  34. Mills, C.E.; Ding, E.; Olsen, B. Protein Purification by Ethanol-Induced Phase Transitions of the Elastin-like Polypeptide (ELP). Ind. Eng. Chem. Res. 2019, 58, 11698–11709. [Google Scholar] [CrossRef]
  35. Darji, S.; Aayush, A.; Estes, K.M.; Strock, J.D.; Thompson, D.H. Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents. Biomacromolecules 2024, 25, 272–281. [Google Scholar] [CrossRef]
  36. Meyer, D.E.; Chilkoti, A. Purification of Recombinant Proteins by Fusion with Thermally-Responsive Polypeptides. Nat. Biotechnol. 1999, 17, 1112–1115. [Google Scholar] [CrossRef] [PubMed]
  37. Meyer, D.E.; Chilkoti, A. Protein Purification by Inverse Transition Cycling. In Protein-Protein Interactions: A Molecular Cloning Manual; Golemis, E.A., Adams, P.D., Eds.; Cold Spring Harbor Laboratory Press: Woodbury, NY, USA, 2002; pp. 329–344. [Google Scholar]
  38. Hassouneh, W.; Christensen, T.; Chilkoti, A. Elastin-like Polypeptides as a Purification Tag for Recombinant Proteins. Curr. Protoc. Protein Sci. 2010, 61, 6–11. [Google Scholar] [CrossRef]
  39. MacEwan, S.R.; Hassouneh, W.; Chilkoti, A. Non-Chromatographic Purification of Recombinant Elastin-like Polypeptides and Their Fusions with Peptides and Proteins from Escherichia coli. J. Vis. Exp. 2014, 88, 51583. [Google Scholar] [CrossRef]
  40. Wang, W.; Wang, Y.; Xia, Z.; Hao, G.; Tuffour, A.; Yan, L.; Chen, J.; Zhu, Y.; Lin, F.; Zhou, Y. Enhancing the Purification and Stability of Superoxide Dismutase by Fusion with Thermoresponsive Self-Assembly of Elastin like Polypeptide. ChemistrySelect 2024, 9, e202401100. [Google Scholar] [CrossRef]
  41. Corich, L.; Busetti, M.; Petix, V.; Passamonti, S.; Bandiera, A. Evaluation of a Biomimetic 3D Substrate Based on the Human Elastin-like Polypeptides (HELPs) Model System for Elastolytic Activity Detection. J. Biotechnol. 2017, 255, 57–65. [Google Scholar] [CrossRef] [PubMed]
  42. Chilkoti, A.; Dreher, M.R.; Meyer, D.E. Design of Thermally Responsive, Recombinant Polypeptide Carriers for Targeted Drug Delivery. Adv. Drug Deliv. Rev. 2002, 54, 1093–1111. [Google Scholar] [CrossRef]
  43. Luan, C.; Parker, T.M.; Prasad, K.U.; Urry, D.W. Differential Scanning Calorimetry Studies of NaCl Effect on the Inverse Temperature Transition of Some Elastin-based Polytetra-, Polypenta-, and Polynonapeptides. Biopolymers 1991, 31, 465–475. [Google Scholar] [CrossRef] [PubMed]
  44. Bandiera, A.; Sist, P.; Terdoslavich, M.; Urbani, U. Phase Transition and Particle Formation of a Human Elastin-like Polypeptide. In Proceedings of the 35th Annual Northeast Bioengineering Conference, Cambridge, MA, USA, 3–5 April 2009; IEEE: New York, NY, USA, 2009; pp. 135–136. [Google Scholar] [CrossRef]
  45. Sist, P.; Bandiera, A.; Urbani, R.; Passamonti, S. Macromolecular and Solution Properties of the Recombinant Fusion Protein HUG. Biomacromolecules 2022, 23, 3336–3348. [Google Scholar] [CrossRef] [PubMed]
  46. Bellingham, C.M.; Woodhouse, K.A.; Robson, P.; Rothstein, S.J.; Keeley, F.W. Self-Aggregation Characteristics of Recombinantly Expressed Human Elastin Polypeptides. Biochim. Biophys. Acta (BBA)-Protein Struct. Mol. Enzymol. 2001, 1550, 6–19. [Google Scholar] [CrossRef]
  47. Reguera, J.; Urry, D.W.; Parker, T.M.; McPherson, D.T.; Rodríguez-Cabello, J.C. Effect of NaCl on the Exothermic and Endothermic Components of the Inverse Temperature Transition of a Model Elastin-like Polymer. Biomacromolecules 2007, 8, 354–358. [Google Scholar] [CrossRef] [PubMed]
  48. Bandiera, A.; Colomina-Alfaro, L.; Sist, P.; Gomez d’Ayala, G.; Zuppardi, F.; Cerruti, P.; Catanzano, O.; Passamonti, S.; Urbani, R. Physicochemical Characterization of a Biomimetic, Elastin-Inspired Polypeptide with Enhanced Thermoresponsive Properties and Improved Cell Adhesion. Biomacromolecules 2023, 24, 5277–5289. [Google Scholar] [CrossRef] [PubMed]
  49. Colomina-Alfaro, L.; Sist, P.; Marchesan, S.; Urbani, R.; Stamboulis, A.; Bandiera, A. A Versatile Elastin-like Carrier for Bioactive Antimicrobial Peptide Production and Delivery. Macromol. Biosci. 2024, 24, 2300236. [Google Scholar] [CrossRef]
  50. Urry, D.W. Physical Chemistry of Biological Free Energy Transduction As Demonstrated by ElasticProtein-Based Polymers. J. Phys. Chem. B 1997, 101, 11007–11028. [Google Scholar] [CrossRef]
  51. Urry, D.W.; Peng, S.; Xu, J.; McPherson, D.T. Characterization of Waters of Hydrophobic Hydration by Microwave Dielectric Relaxation. J. Am. Chem. Soc. 1997, 119, 1161–1162. [Google Scholar] [CrossRef]
  52. Reiersen, H.; Clarke, A.R.; Rees, A.R. Short Elastin-like Peptides Exhibit the Same Temperature-Induced Structural Transitions as Elastin Polymers: Implications for Protein Engineering. J. Mol. Biol. 1998, 283, 255–264. [Google Scholar] [CrossRef]
  53. Nuhn, H.; Klok, H.A. Secondary Structure Formation and LCST Behavior of Short Elastin-like Peptides. Biomacromolecules 2008, 9, 2755–2763. [Google Scholar] [CrossRef] [PubMed]
  54. Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M.R.; Appel, R.D.; Bairoch, A. Protein Identification and Analysis Tools on the ExPASy Server. In The Proteomics Protocols Handbook; Humana Press: Totowa, NJ, USA, 2005; pp. 571–607. [Google Scholar]
  55. Garnier, J.; Gibrat, J.-F.; Robson, B. [32] GOR Method for Predicting Protein Secondary Structure from Amino Acid Sequence. In Methods in Enzymology; Elsevier: Amsterdam, The Netherlands, 1996; Volume 266, pp. 540–553. ISBN 0076-6879. [Google Scholar]
  56. Berne, B.J.; Pecora, R. Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics; Dover Publications, Ed.; Unabridged; Courier Corporation: Washington, DC, USA, 2000. [Google Scholar]
  57. Zhang, Y.; Gao, S.; Qi, X.; Zhu, S.; Xu, S.; Liang, Y.; Kong, F.; Yang, S.; Wang, R.; Wang, Y.; et al. Novel Biocatalytic Strategy of Levan: His-ELP-Intein-Tagged Protein Purification and Biomimetic Mineralization. Carbohydr. Polym. 2022, 288, 119398. [Google Scholar] [CrossRef] [PubMed]
  58. Phan, H.T.; Conrad, U. Membrane-Based Inverse Transition Cycling: An Improved Means for Purifying Plant-Derived Recombinant Protein-Elastin-like Polypeptide Fusions. Int. J. Mol. Sci. 2011, 12, 2808–2821. [Google Scholar] [CrossRef] [PubMed]
  59. Leonard, A.; Koria, P. Growth Factor Functionalized Biomaterial for Drug Delivery and Tissue Regeneration. J. Bioact. Compat. Polym. 2017, 32, 568–581. [Google Scholar] [CrossRef] [PubMed]
  60. Sarvestani, R.; Latifi, A.M.; Alizadeh, H.; Mirzaei, M. An Approach for Recombinant Epidermal Growth Factor Purification Using an Elastin-like Protein Tag. J. Appl. Biotechnol. Rep. 2021, 8, 127–132. [Google Scholar] [CrossRef]
  61. Lee, K.M.; Jung, G.S.; Park, J.K.; Choi, S.K.; Jeon, W.B. Effects of Arg-Gly-Asp-Modified Elastin-like Polypeptide on Pseudoislet Formation via up-Regulation of Cell Adhesion Molecules and Extracellular Matrix Proteins. Acta Biomater. 2013, 9, 5600–5608. [Google Scholar] [CrossRef]
  62. Hwang, Y.J.; Jung, G.S.; Jeon, W.B.; Lee, K.M. Arg-Gly-Asp-Modified Elastin-like Polypeptide Regulates Cell Proliferation and Cell Cycle Proteins via the Phosphorylation of Erk and Akt in Pancreatic β-Cell. Heliyon 2020, 6, e04918. [Google Scholar] [CrossRef] [PubMed]
  63. Liu, Y.; Gilchrist, A.E.; Heilshorn, S.C. Engineered Protein Hydrogels as Biomimetic Cellular Scaffolds. Adv. Mater. 2024, 36, 2407794. [Google Scholar] [CrossRef] [PubMed]
  64. Colomina-Alfaro, L.; Sist, P.; D’Andrea, P.; Urbani, R.; Marchesan, S.; Stamboulis, A.; Bandiera, A. Materials Derived from the Human Elastin-like Polypeptide Fusion with an Antimicrobial Peptide Strongly Promote Cell Adhesion. J. Mater. Chem. B 2024. [Google Scholar] [CrossRef] [PubMed]
  65. Hu, F.; Ke, T.; Li, X.; Mao, P.H.; Jin, X.; Hui, F.L.; Ma, X.D.; Ma, L.X. Expression and Purification of an Antimicrobial Peptide by Fusion with Elastin-like Polypeptides in Escherichia coli. Appl. Biochem. Biotechnol. 2010, 160, 2377–2387. [Google Scholar] [CrossRef]
  66. Atefyekta, S.; Pihl, M.; Lindsay, C.; Heilshorn, S.C.; Andersson, M. Antibiofilm Elastin-like Polypeptide Coatings: Functionality, Stability, and Selectivity. Acta Biomater. 2019, 83, 245–256. [Google Scholar] [CrossRef]
  67. Shitashima, Y.; Shimozawa, T.; Kumagai, A.; Miyawaki, A.; Asahi, T. Two Distinct Fluorescence States of the Ligand-Induced Green Fluorescent Protein UnaG. Biophys. J. 2017, 113, 2805–2814. [Google Scholar] [CrossRef] [PubMed]
  68. Yeh, J.T.H.; Nam, K.; Yeh, J.T.H.; Perrimon, N. EUnaG: A New Ligand-Inducible Fluorescent Reporter to Detect Drug Transporter Activity in Live Cells. Sci. Rep. 2017, 7, 41619. [Google Scholar] [CrossRef]
  69. Erapaneedi, R.; Belousov, V.V.; Schäfers, M.; Kiefer, F. A Novel Family of Fluorescent Hypoxia Sensors Reveal Strong Heterogeneity in Tumor Hypoxia at the Cellular Level. EMBO J. 2016, 35, 102–113. [Google Scholar] [CrossRef] [PubMed]
  70. Uda, Y.; Goto, Y.; Oda, S.; Kohchi, T.; Matsuda, M.; Aoki, K. Efficient Synthesis of Phycocyanobilin in Mammalian Cells for Optogenetic Control of Cell Signaling. Proc. Natl. Acad. Sci. USA 2017, 114, 11962–11967. [Google Scholar] [CrossRef] [PubMed]
  71. Iwatani, S.; Yamana, K.; Nakamura, H.; Nishida, K.; Morisawa, T.; Mizobuchi, M.; Osawa, K.; Iijima, K.; Morioka, I. A Novel Method for Measuring Serum Unbound Bilirubin Levels Using Glucose Oxidase–Peroxidase and Bilirubin-Inducible Fluorescent Protein (UnaG): No Influence of Direct Bilirubin. Int. J. Mol. Sci. 2020, 21, 6778. [Google Scholar] [CrossRef] [PubMed]
  72. Tien Tai, T.; Adachi, Y.; Taketani, S. A Fluorescence-Based Quantitative Analysis for Total Bilirubin in Blood and Urine. Lab. Med. 2022, 53, 6–11. [Google Scholar] [CrossRef] [PubMed]
  73. Wu, J.; Moriwaki, K.; Asuka, T.; Nakai, R.; Kanda, S.; Taniguchi, M.; Sugiyama, T.; Shin-Ichiro, Y.; Kunii, M.; Nagasawa, T.; et al. EHBP1L1, an Apicobasal Polarity Regulator, Is Critical for Nuclear Polarization during Enucleation of Erythroblasts. Blood Adv. 2023, 7, 3382–3394. [Google Scholar] [CrossRef] [PubMed]
  74. Al Reza, H.; Farooqui, Z.; Al Reza, A.; Conroy, C.; Iwasawa, K.; Ogura, Y.; Okita, K.; Osafune, K.; Takebe, T. Synthetic Augmentation of Bilirubin Metabolism in Human Pluripotent Stem Cell-Derived Liver Organoids. Stem Cell Rep. 2023, 18, 2071–2083. [Google Scholar] [CrossRef]
  75. Fang, B.; Card, P.D.; Chen, J.; Li, L.; Laughlin, T.; Jarrold, B.; Zhao, W.; Benham, A.M.; Määttä, A.T.; Hawkins, T.J.; et al. A Potential Role of Keratinocyte-Derived Bilirubin in Human Skin Yellowness and Its Amelioration by Sucrose Laurate/Dilaurate. Int. J. Mol. Sci. 2022, 23, 5884. [Google Scholar] [CrossRef]
  76. Iwatani, S.; Nakamura, H.; Kurokawa, D.; Yamana, K.; Nishida, K.; Fukushima, S.; Koda, T.; Nishimura, N.; Nishio, H.; Iijima, K.; et al. Fluorescent Protein-Based Detection of Unconjugated Bilirubin in Newborn Serum. Sci. Rep. 2016, 6, 28489. [Google Scholar] [CrossRef] [PubMed]
  77. Takeda, T.A.; Mu, A.; Tai, T.T.; Kitajima, S.; Taketani, S. Continuous de Novo Biosynthesis of Haem and Its Rapid Turnover to Bilirubin Are Necessary for Cytoprotection against Cell Damage. Sci. Rep. 2015, 5, 10488. [Google Scholar] [CrossRef] [PubMed]
  78. Kono, Y.; Ishizawa, T.; Kokudo, N.; Kuriki, Y.; Iwatate, R.J.; Kamiya, M.; Urano, Y.; Kumagai, A.; Kurokawa, H.; Miyawaki, A.; et al. On-Site Monitoring of Postoperative Bile Leakage Using Bilirubin-Inducible Fluorescent Protein. World J. Surg. 2020, 44, 4245–4253. [Google Scholar] [CrossRef] [PubMed]
  79. Adeosun, S.; Moore, K.; Lang, D.; Nwaneri, A.; Hinds, T., Jr.; Stec, D. A Novel Fluorescence-Based Assay for the Measurement of Biliverdin Reductase Activity. React. Oxyg. Species 2018, 5, 35–45. [Google Scholar] [CrossRef]
  80. Ishikawa, K.; Kodama, Y. Bilirubin Distribution in Plants at the Subcellular and Tissue Levels. Plant Cell Physiol. 2024, 65, 762–769. [Google Scholar] [CrossRef] [PubMed]
  81. Kataura, T.; Saiki, S.; Ishikawa, K.I.; Akamatsu, W.; Sasazawa, Y.; Hattori, N.; Imoto, M. BRUP-1, an Intracellular Bilirubin Modulator, Exerts Neuroprotective Activity in a Cellular Parkinson’s Disease Model. J. Neurochem. 2020, 155, 81–97. [Google Scholar] [CrossRef] [PubMed]
  82. Park, J.S.; Nam, E.; Lee, H.K.; Lim, M.H.; Rhee, H.W. In Cellulo Mapping of Subcellular Localized Bilirubin. ACS Chem. Biol. 2016, 11, 2177–2185. [Google Scholar] [CrossRef] [PubMed]
  83. Navarro, R.; Chen, L.C.; Rakhit, R.; Wandless, T.J. A Novel Destabilizing Domain Based on a Small-Molecule Dependent Fluorophore. ACS Chem. Biol. 2016, 11, 2101–2104. [Google Scholar] [CrossRef] [PubMed]
  84. Vasavda, C.; Kothari, R.; Malla, A.P.; Tokhunts, R.; Lin, A.; Ji, M.; Ricco, C.; Xu, R.; Saavedra, H.G.; Sbodio, J.I.; et al. Bilirubin Links Heme Metabolism to Neuroprotection by Scavenging Superoxide. Cell Chem. Biol. 2019, 26, 1450–1460.e7. [Google Scholar] [CrossRef] [PubMed]
  85. Ko, S.; Kwon, J.; Shim, S.H. Enhanced UnaG with Minimal Labeling Artifact for Single-Molecule Localization Microscopy. Front. Mol. Biosci. 2021, 8, 647590. [Google Scholar] [CrossRef] [PubMed]
  86. Shum, M.; Shintre, C.A.; Althoff, T.; Gutierrez, V.; Segawa, M.; Saxberg, A.D.; Martinez, M.; Adamson, R.; Young, M.R.; Faust, B.; et al. ABCB10 Exports Mitochondrial Biliverdin, Driving Metabolic Maladaptation in Obesity. Sci. Transl. Med. 2021, 13, eabd1869. [Google Scholar] [CrossRef]
  87. Ruskowitz, E.R.; Munoz-Robles, B.G.; Strange, A.C.; Butcher, C.H.; Kurniawan, S.; Filteau, J.R.; DeForest, C.A. Spatiotemporal Functional Assembly of Split Protein Pairs through a Light-Activated SpyLigation. Nat. Chem. 2023, 15, 694–704. [Google Scholar] [CrossRef]
  88. Yan, D.; Wu, X.T.; Chen, X.; Wang, J.; Ge, F.; Wu, M.; Wu, J.; Zhang, N.; Xiao, M.; Wu, X.; et al. Maternal Linoleic Acid-Rich Diet Ameliorates Bilirubin Neurotoxicity in Offspring Mice. Cell Death Discov. 2024, 10, 329. [Google Scholar] [CrossRef]
  89. Uchida, Y.; Takahashi, Y.; Kurata, C.; Morimoto, Y.; Ohtani, E.; Tosaki, A.; Kumagai, A.; Greimel, P.; Nishikubo, T.; Miyawaki, A. Urinary Lumirubin Excretion in Jaundiced Preterm Neonates during Phototherapy with Blue Light-Emitting Diode vs. Green Fluorescent Lamp. Sci. Rep. 2023, 13, 18359. [Google Scholar] [CrossRef] [PubMed]
  90. Uchida, Y.; Takahashi, Y.; Morimoto, Y.; Greimel, P.; Tosaki, A.; Kumagai, A.; Nishikubo, T.; Miyawaki, A. Noninvasive Monitoring of Bilirubin Photoisomer Excretion during Phototherapy. Sci. Rep. 2022, 12, 11798. [Google Scholar] [CrossRef]
  91. Chia, H.E.; Zuo, T.; Koropatkin, N.M.; Marsh, E.N.G.; Biteen, J.S. Imaging Living Obligate Anaerobic Bacteria with Bilin-Binding Fluorescent Proteins. Curr. Res. Microb. Sci. 2020, 1, 1–6. [Google Scholar] [CrossRef] [PubMed]
  92. Chia, H.E.; Koebke, K.J.; Rangarajan, A.A.; Koropatkin, N.M.; Marsh, E.N.G.; Biteen, J.S. New Orange Ligand-Dependent Fluorescent Reporter for Anaerobic Imaging. ACS Chem. Biol. 2021, 16, 2109–2115. [Google Scholar] [CrossRef] [PubMed]
  93. Ishchuk, O.P.; Frost, A.T.; Muñiz-Paredes, F.; Matsumoto, S.; Laforge, N.; Eriksson, N.L.; Martínez, J.L.; Petranovic, D. Improved Production of Human Hemoglobin in Yeast by Engineering Hemoglobin Degradation. Metab. Eng. 2021, 66, 259–267. [Google Scholar] [CrossRef] [PubMed]
  94. Tchagang, C.F.; Mah, T.F.; Campbell-Valois, F.X. Anaerobic Fluorescent Reporters for Live Imaging of Pseudomonas Aeruginosa. Front. Microbiol. 2023, 14, 1245755. [Google Scholar] [CrossRef]
  95. Richard, C.S.M.; Dey, H.; Øyen, F.; Maqsood, M.; Blencke, H.M. Outer Membrane Integrity-Dependent Fluorescence of the Japanese Eel UnaG Protein in Live Escherichia coli Cells. Biosensors 2023, 13, 232. [Google Scholar] [CrossRef] [PubMed]
  96. Banerjee, B.; Olajide, O.J.; Bortolussi, G.; Muro, A.F. Activation of Alternative Bilirubin Clearance Pathways Partially Reduces Hyperbilirubinemia in a Mouse Model Lacking Functional Ugt1a1 Activity. Int. J. Mol. Sci. 2022, 23, 10703. [Google Scholar] [CrossRef]
  97. Liu, H.W.; Lai, K.; Gong, L.N.; Shi, H.B.; Yin, S.K.; Wang, L.Y. Measuring Endogenous Levels of Unconjugated Bilirubin Released from Isolated Murine Brain Tissue during Oxygen-Glucose Deprivation. STAR Protoc. 2023, 4, 102550. [Google Scholar] [CrossRef]
  98. Zahradník, J.; Dey, D.; Marciano, S.; Kolářová, L.; Charendoff, C.I.; Subtil, A.; Schreiber, G. A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets. ACS Synth. Biol. 2021, 10, 3445–3460. [Google Scholar] [CrossRef]
  99. To, T.L.; Zhang, Q.; Shu, X. Structure-Guided Design of a Reversible Fluorogenic Reporter of Protein-Protein Interactions. Protein Sci. 2016, 25, 748–753. [Google Scholar] [CrossRef]
  100. Eaton, H.E.; Kobayashi, T.; Dermody, T.S.; Johnston, R.N.; Jais, P.H.; Shmulevitz, M.; López, S. African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions. J. Virol. 2017, 91, e02416-16. [Google Scholar] [CrossRef] [PubMed]
  101. Zeng, Z.; Huang, B.; Huang, S.; Zhang, R.; Yan, S.; Yu, X.; Shu, Y.; Zhao, C.; Lei, J.; Zhang, W.; et al. The Development of a Sensitive Fluorescent Protein-Based Transcript Reporter for High Throughput Screening of Negative Modulators of LncRNAs. Genes. Dis. 2018, 5, 62–74. [Google Scholar] [CrossRef] [PubMed]
  102. Kim, H.; Ellis, V.D.; Woodman, A.; Zhao, Y.; Arnold, J.J.; Cameron, C.E. RNA-Dependent RNA Polymerase Speed and Fidelity Are Not the Only Determinants of the Mechanism or Efficiency of Recombination. Genes 2019, 10, 968. [Google Scholar] [CrossRef]
  103. Philip, A.A.; Perry, J.L.; Eaton, H.E.; Shmulevitz, M.; Hyser, J.M.; Patton, J.T. Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System. J. Virol. 2019, 93, e01616-19. [Google Scholar] [CrossRef] [PubMed]
  104. Liu, Y.; Zhang, H.; Chen, R.; Wu, Y.; Yang, X.; Liu, X.; Zeng, S.; Guo, W. UnaG as a Reporter in Adeno-Associated Virus-Mediated Gene Transfer for Biomedical Imaging. J. Biophotonics 2020, 13, e202000182. [Google Scholar] [CrossRef] [PubMed]
  105. Gan, C.; Cui, J.; Zhang, W.; Wang, Y.; Huang, A.; Hu, J. DNA Engineering and Hepatitis B Virus Replication. Front. Microbiol. 2021, 12, 783040. [Google Scholar] [CrossRef] [PubMed]
  106. Sam, M.; Selman, M.; Zhao, W.; Jung, J.; Willingham, A.; Phan, U.; Starling, G.C.; Gao, Q. Engineering Oncolytic Coxsackievirus A21 with Small Transgenes and Enabling Cell-Mediated Virus Delivery by Integrating Viral CDNA into the Genome. J. Virol. 2023, 97, e00309-23. [Google Scholar] [CrossRef]
  107. Okuwa, T.; Himeda, T.; Utani, K.; Higuchi, M. Generation of a Recombinant Saffold Virus Expressing UnaG as a Marker for the Visualization of Viral Infection. Virol. J. 2023, 20, 175. [Google Scholar] [CrossRef]
  108. Yang, J.; Xiao, Y.; Lidsky, P.V.; Wu, C.T.; Bonser, L.R.; Peng, S.; Garcia-Knight, M.A.; Tassetto, M.; Chung, C.I.; Li, X.; et al. Fluorogenic Reporter Enables Identification of Compounds That Inhibit SARS-CoV-2. Nat. Microbiol. 2023, 8, 121–134. [Google Scholar] [CrossRef] [PubMed]
  109. Schmitz, C.; Pepelanova, I.; Seliktar, D.; Potekhina, E.; Belousov, V.V.; Scheper, T.; Lavrentieva, A. Live Reporting for Hypoxia: Hypoxia Sensor–Modified Mesenchymal Stem Cells as in Vitro Reporters. Biotechnol. Bioeng. 2020, 117, 3265–3276. [Google Scholar] [CrossRef] [PubMed]
  110. Yousaf, I.; Kaeppler, J.; Frost, S.; Seymour, L.W.; Jacobus, E.J. Attenuation of the Hypoxia Inducible Factor Pathway after Oncolytic Adenovirus Infection Coincides with Decreased Vessel Perfusion. Cancers 2020, 12, 851. [Google Scholar] [CrossRef] [PubMed]
  111. Panicucci, G.; Iacopino, S.; De Meo, E.; Perata, P.; Weits, D.A. An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue. Biosensors 2020, 10, 197. [Google Scholar] [CrossRef] [PubMed]
  112. Schmitz, C.; Potekhina, E.; Belousov, V.V.; Lavrentieva, A. Hypoxia Onset in Mesenchymal Stem Cell Spheroids: Monitoring with Hypoxia Reporter Cells. Front. Bioeng. Biotechnol. 2021, 9, 611837. [Google Scholar] [CrossRef] [PubMed]
  113. Sattiraju, A.; Kang, S.; Giotti, B.; Chen, Z.; Marallano, V.J.; Brusco, C.; Ramakrishnan, A.; Shen, L.; Tsankov, A.M.; Hambardzumyan, D.; et al. Hypoxic Niches Attract and Sequester Tumor-Associated Macrophages and Cytotoxic T Cells and Reprogram Them for Immunosuppression. Immunity 2023, 56, 1825–1843.e6. [Google Scholar] [CrossRef] [PubMed]
  114. Lavilla-Puerta, M.; Giuntoli, B. Assessing In Vivo Oxygen Dynamics Using Plant N-Terminal Degrons in Saccharomyces cerevisiae. In Fluorescent Proteins; Sharma, M., Ed.; Humana: New York, NY, USA, 2023; pp. 269–286. [Google Scholar]
  115. Dienemann, S.; Schmidt, V.; Fleischhammer, T.; Mueller, J.H.; Lavrentieva, A. Comparative Analysis of Hypoxic Response of Human Microvascular and Umbilical Vein Endothelial Cells in 2D and 3D Cell Culture Systems. J. Cell. Physiol. 2023, 238, 1111–1120. [Google Scholar] [CrossRef]
  116. Bauer, N.; Maisuls, I.; Pereira da Graça, A.; Reinhardt, D.; Erapaneedi, R.; Kirschnick, N.; Schäfers, M.; Grashoff, C.; Landfester, K.; Vestweber, D.; et al. Genetically Encoded Dual Fluorophore Reporters for Graded Oxygen-Sensing in Light Microscopy. Biosens. Bioelectron. 2023, 221, 114917. [Google Scholar] [CrossRef] [PubMed]
  117. Ishiyama, R.; Yoshida, K.; Oikawa, K.; Takai-Todaka, R.; Kato, A.; Kanamori, K.; Nakanishi, A.; Haga, K.; Katayama, K. Production of Infectious Reporter Murine Norovirus by VP2 Trans-Complementation. J. Virol. 2024, 98, e01261-23. [Google Scholar] [CrossRef]
  118. Bauer, N.; Kiefer, F. Genetically Encoded Reporters to Monitor Hypoxia. In Hypoxia; Gilkes, D.M., Ed.; Humana: New York, NY, USA, 2024; Volume 2755, pp. 3–29. [Google Scholar]
  119. Fleischhammer, T.M.; Dienemann, S.; Ulber, N.; Pepelanova, I.; Lavrentieva, A. Detection of Hypoxia in 2D and 3D Cell Culture Systems Using Genetically Encoded Fluorescent Hypoxia Sensors. In Hypoxia; Gilkes, D.M., Ed.; Humana: New York, NY, USA, 2024; Volume 2755, pp. 31–48. [Google Scholar]
  120. Kwon, J.; Park, J.S.; Kang, M.; Choi, S.; Park, J.; Kim, G.T.; Lee, C.; Cha, S.; Rhee, H.W.; Shim, S.H. Bright Ligand-Activatable Fluorescent Protein for High-Quality Multicolor Live-Cell Super-Resolution Microscopy. Nat. Commun. 2020, 11, 273. [Google Scholar] [CrossRef] [PubMed]
  121. Cao, X.; Zhang, C.; Gao, Z.; Liu, Y.; Zhao, Y.; Yang, Y.; Chen, J.; Jimenez, R.; Xu, J. Ultrafast Internal Conversion Dynamics of Bilirubin Bound to UnaG and Its N57A Mutant. Phys. Chem. Chem. Phys. 2019, 21, 2365–2371. [Google Scholar] [CrossRef] [PubMed]
  122. Asad, M.; Laurent, A.D. Exploring Structural Dynamics and Optical Properties of UnaG Fluorescent Protein upon N57 Mutations. Phys. Chem. Chem. Phys. 2022, 24, 3816–3825. [Google Scholar] [CrossRef] [PubMed]
  123. Eczacioglu, N.; Ulusu, Y.; Gokce, İ.; Lakey, J.H. Investigation of Mutations (L41F, F17M, N57E, Y99F_Y134W) Effects on the TolAIII-UnaG Fluorescence Protein’s Unconjugated Bilirubin (UC-BR) Binding Ability and Thermal Stability Properties. Prep. Biochem. Biotechnol. 2022, 52, 365–374. [Google Scholar] [CrossRef] [PubMed]
  124. Sist, P.; Saeed, S.; Tramer, F.; Bandiera, A.; Passamonti, S. Standardized Lab-Scale Production of the Recombinant Fusion Protein HUG for the Nanoscale Analysis of Bilirubin. MethodsX 2024, 13, 103001. [Google Scholar] [CrossRef] [PubMed]
  125. Sist, P.; Tramer, F.; Urbani, R.; Bandiera, A.; Passamonti, S. Preparation and Validation of Nanomolar Aqueous Bilirubin Standard Solutions. MethodsX 2025, 14, 103123. [Google Scholar] [CrossRef]
  126. Sist, P.; Tramer, F.; Bandiera, A.; Urbani, R.; Redenšek Trampuž, S.; Dolžan, V.; Passamonti, S. Nanoscale Bilirubin Analysis in Translational Research and Precision Medicine by the Recombinant Protein HUG. Int. J. Mol. Sci. 2023, 24, 16289. [Google Scholar] [CrossRef]
  127. Tramer, F.; Sist, P.; Cardenas-Perez, R.; Urbani, R.; Bortolussi, G.; Passamonti, S. Combined Fluorometric Analysis of Biliverdin and Bilirubin by the Recombinant Protein HUG. MethodsX 2024, 13, 102979. [Google Scholar] [CrossRef] [PubMed]
  128. Pelizzo, P.; Stebel, M.; Medic, N.; Sist, P.; Vanzo, A.; Anesi, A.; Vrhovsek, U.; Tramer, F.; Passamonti, S. Cyanidin 3-Glucoside Targets a Hepatic Bilirubin Transporter in Rats. Biomed. Pharmacother. 2023, 157, 114044. [Google Scholar] [CrossRef] [PubMed]
  129. Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; et al. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596, 583–589. [Google Scholar] [CrossRef]
  130. Chen, R.F. The Fluorescence of Bilirubin-Albumin Complexes. In Fluorescence Techniques in Cell Biology; Springer: Berlin/Heidelberg, Germany, 1973; pp. 273–282. [Google Scholar]
  131. Tatikolov, A.S.; Pronkin, P.G.; Panova, I.G. Bilirubin: Photophysical and Photochemical Properties, Phototherapy, Analytical Methods of Measurement. A Short Review. Biophys. Chem. 2025, 318, 107378. [Google Scholar] [CrossRef] [PubMed]
  132. Tonelotto, V.; Costa-Garcia, M.; O’Reilly, E.; Smith, K.F.; Slater, K.; Dillon, E.T.; Pendino, M.; Higgins, C.; Sist, P.; Bosch, R.; et al. 1,4-Dihydroxy Quininib Activates Ferroptosis Pathways in Metastatic Uveal Melanoma and Reveals a Novel Prognostic Biomarker Signature. Cell Death Discov. 2024, 10, 70. [Google Scholar] [CrossRef] [PubMed]
  133. Voltarelli, V.A.; Alves de Souza, R.W.; Miyauchi, K.; Hauser, C.J.; Otterbein, L.E. Heme: The Lord of the Iron Ring. Antioxidants 2023, 12, 1074. [Google Scholar] [CrossRef] [PubMed]
  134. Levitt, D.G.; Levitt, M.D. Quantitative Assessment of the Multiple Processes Responsible for Bilirubin Homeostasis in Health and Disease. Clin. Exp. Gastroenterol. 2014, 7, 307–328. [Google Scholar] [CrossRef]
  135. Ryter, S.W.; Tyrrell, R.M. The Heme Synthesis and Degradation Pathways: Role in Oxidant Sensitivity. Free Radic. Biol. Med. 2000, 28, 289–309. [Google Scholar] [CrossRef]
  136. Martelanc, M.; Žiberna, L.; Passamonti, S.; Franko, M. Direct Determination of Free Bilirubin in Serum at Sub-Nanomolar Levels. Anal. Chim. Acta 2014, 809, 174–182. [Google Scholar] [CrossRef]
  137. Dunaway, L.S.; Loeb, S.A.; Petrillo, S.; Tolosano, E.; Isakson, B.E. Heme Metabolism in Nonerythroid Cells. J. Biol. Chem. 2024, 300, 107132. [Google Scholar] [CrossRef]
  138. Ryter, S.W.; Alam, J.; Choi, A.M.K. Heme Oxygenase-1/Carbon Monoxide: From Basic Science to Therapeutic Applications. Physiol. Rev. 2006, 86, 583–650. [Google Scholar] [CrossRef] [PubMed]
  139. Costa Silva, R.C.M.; Correa, L.H.T. Heme Oxygenase 1 in Vertebrates: Friend and Foe. Cell Biochem. Biophys. 2022, 80, 97–113. [Google Scholar] [CrossRef]
  140. Čvorović, J.; Passamonti, S. Membrane Transporters for Bilirubin and Its Conjugates: A Systematic Review. Front. Pharmacol. 2017, 8, 887. [Google Scholar] [CrossRef] [PubMed]
  141. Gåfvels, M.; Holmström, P.; Somell, A.; Sjövall, F.; Svensson, J.O.; Ståhle, L.; Broomé, U.; Stål, P. A Novel Mutation in the Biliverdin Reductase-A Gene Combined with Liver Cirrhosis Results in Hyperbiliverdinaemia (Green Jaundice). Liver Int. 2009, 29, 1116–1124. [Google Scholar] [CrossRef] [PubMed]
  142. Bai, W.; Huo, S.; Zhou, G.; Li, J.; Yang, Y.; Shao, J. Biliverdin Modulates the Nrf2/A20/EEF1A2 Axis to Alleviate Cerebral Ischemia-Reperfusion Injury by Inhibiting Pyroptosis. Biomed. Pharmacother. 2023, 165, 115057. [Google Scholar] [CrossRef] [PubMed]
  143. Bortolussi, G.; Shi, X.; Ten Bloemendaal, L.; Banerjee, B.; De Waart, D.R.; Baj, G.; Chen, W.; Oude Elferink, R.P.; Beuers, U.; Paulusma, C.C.; et al. Long-Term Effects of Biliverdin Reductase a Deficiency in Ugt1−/− Mice: Impact on Redox Status and Metabolism. Antioxidants 2021, 10, 2029. [Google Scholar] [CrossRef] [PubMed]
  144. Yao, Z.; Xue, K.; Chen, J.; Zhang, Y.; Zhang, G.; Zheng, Z.; Li, Z.; Li, Z.; Wang, F.; Sun, X.; et al. Biliverdin Improved Angiogenesis and Suppressed Apoptosis via PI3K/Akt-Mediated Nrf2 Antioxidant System to Promote Ischemic Flap Survival. Free Radic. Biol. Med. 2024, 225, 35–52. [Google Scholar] [CrossRef] [PubMed]
  145. Wegiel, B.; Otterbein, L.E. Go Green: The Anti-Inflammatory Effects of Biliverdin Reductase. Front. Pharmacol. 2012, 3, 47. [Google Scholar] [CrossRef] [PubMed]
  146. Schwertner, H.A.; Jackson, W.G.; Tolan, G. Association of Low Serum Concentration of Bilirubin with Increased Risk of Coronary Artery Disease. Clin. Chem. 1994, 40, 18–23. [Google Scholar] [CrossRef] [PubMed]
  147. Zuo, L.; Huang, J.; Zhang, H.; Huang, B.; Wu, X.; Chen, L.; Xia, S.; Dong, X.; Hao, G. Dose-Response Association Between Bilirubin and Cardiovascular Disease: A Systematic Review and Meta-Analysis. Angiology 2022, 73, 911–919. [Google Scholar] [CrossRef]
  148. Wang, G.; Qiao, L.; Tang, Z.; Zhou, S.; Min, J.; Li, M. Association between Bilirubin Levels and Risk of Stroke: A Systematic Review and Meta-Analysis. BMJ Open 2023, 13, e064433. [Google Scholar] [CrossRef] [PubMed]
  149. Nikouei, M.; Cheraghi, M.; Ghaempanah, F.; Kohneposhi, P.; Saniee, N.; Hemmatpour, S.; Moradi, Y. The Association between Bilirubin Levels, and the Incidence of Metabolic Syndrome and Diabetes Mellitus: A Systematic Review and Meta-Analysis of Cohort Studies. Clin. Diabetes Endocrinol. 2024, 10, 1. [Google Scholar] [CrossRef]
  150. Huang, S.-S.; Ding, Y.; Yi, X.-N.; Mao, H.-Y.; Xie, Z.-Y.; Shen, X.-K.; Lu, Y.; Yan, J.; Wang, Y.-W.; Yang, Z.-X. Exploring the Inverse Relationship between Serum Total Bilirubin and Systemic Immune-Inflammation Index: Insights from NHANES Data (2009–2018). Eur. J. Med. Res. 2024, 29, 362. [Google Scholar] [CrossRef] [PubMed]
  151. Liang, C.; Yu, Z.; Bai, L.; Hou, W.; Tang, S.; Zhang, W.; Chen, X.; Hu, Z.; Duan, Z.; Zheng, S.; et al. Association of Serum Bilirubin with Metabolic Syndrome and Non-Alcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis. Front. Endocrinol. 2022, 13, 869579. [Google Scholar] [CrossRef] [PubMed]
  152. Marconi, V.C.; Duncan, M.S.; So-Armah, K.; Re, V.L.; Lim, J.K.; Butt, A.A.; Goetz, M.B.; Rodriguez-Barradas, M.C.; Alcorn, C.W.; Lennox, J.; et al. Bilirubin Is Inversely Associated with Cardiovascular Disease Among HIV-Positive and HIV-Negative Individuals in VACS (Veterans Aging Cohort Study). J. Am. Heart Assoc. 2018, 7, e007792. [Google Scholar] [CrossRef] [PubMed]
  153. Bosma, P.J.; Seppen, J.; Goldhoorn, B.; Bakker, C.; Elferink, R.P.J.O.; Chowdhury, J.R.; Chowdhuryl, N.R.; Jansen, P.L.M. Bilirubin UDP-Glucuronosyltransferase 1 Is the Only Relevant Bilirubin Glucuronidating Isoform in Man. J. Biol. Chem. 1994, 269, 17960–17964. [Google Scholar] [CrossRef] [PubMed]
  154. Chen, Z.; Su, D.; Ai, L.; Jiang, X.; Wu, C.; Xu, Q.; Wang, X.; Fan, Z. UGT1A1 Sequence Variants Associated with Risk of Adult Hyperbilirubinemia: A Quantitative Analysis. Gene 2014, 552, 32–38. [Google Scholar] [CrossRef] [PubMed]
  155. Maruhashi, T.; Soga, J.; Fujimura, N.; Idei, N.; Mikami, S.; Iwamoto, Y.; Kajikawa, M.; Matsumoto, T.; Kihara, Y.; Chayama, K.; et al. Hyperbilirubinemia, Augmentation of Endothelial Function, and Decrease in Oxidative Stress in Gilbert Syndrome. Circulation 2012, 126, 598–603. [Google Scholar] [CrossRef]
  156. Horsfall, L.J.; Nazareth, I.; Petersen, I. Cardiovascular Events as a Function of Serum Bilirubin Levels in a Large, Statin-Treated Cohort. Circulation 2012, 126, 2556–2564. [Google Scholar] [CrossRef]
  157. Bulmer, A.C.; Verkade, H.J.; Wagner, K.-H. Bilirubin and beyond: A Review of Lipid Status in Gilbert’s Syndrome and Its Relevance to Cardiovascular Disease Protection. Prog. Lipid Res. 2013, 52, 193–205. [Google Scholar] [CrossRef] [PubMed]
  158. Mölzer, C.; Wallner, M.; Kern, C.; Tosevska, A.; Zadnikar, R.; Doberer, D.; Marculescu, R.; Wagner, K.-H. Characteristics of the Heme Catabolic Pathway in Mild Unconjugated Hyperbilirubinemia and Their Associations with Inflammation and Disease Prevention. Sci. Rep. 2017, 7, 755. [Google Scholar] [CrossRef] [PubMed]
  159. Zinellu, A.; Mangoni, A.A. The Role of Bilirubin as a Biomarker of Rheumatic Diseases: A Systematic Review and Meta-Analysis. Front. Immunol. 2024, 15, 1369284. [Google Scholar] [CrossRef] [PubMed]
  160. Horsfall, L.J.; Nazareth, I.; Pereira, S.P.; Petersen, I. Gilbert’s Syndrome and the Risk of Death: A Population-Based Cohort Study. J. Gastroenterol. Hepatol. 2013, 28, 1643–1647. [Google Scholar] [CrossRef]
  161. Farrera, J.-A.; Jaumà, A.; Ribó, J.M.; Asunción Peiré, M.; Parellada, P.P.; Roques-Choua, S.; Bienvenue, E.; Seta, P. The Antioxidant Role of Bile Pigments Evaluated by Chemical Tests. Bioorg Med. Chem. 1994, 2, 181–185. [Google Scholar] [CrossRef]
  162. Vera, T.; Granger, J.P.; Stec, D.E. Inhibition of Bilirubin Metabolism Induces Moderate Hyperbilirubinemia and Attenuates ANG II-Dependent Hypertension in Mice. Am. J. Physiol.-Regul. Integr. Comp. Physiol. 2009, 297, R738–R743. [Google Scholar] [CrossRef] [PubMed]
  163. Jansen, T.; Daiber, A. Direct Antioxidant Properties of Bilirubin and Biliverdin. Is There a Role for Biliverdin Reductase? Front. Pharmacol. 2012, 3, 30. [Google Scholar] [CrossRef] [PubMed]
  164. Ziberna, L.; Martelanc, M.; Franko, M.; Passamonti, S. Bilirubin Is an Endogenous Antioxidant in Human Vascular Endothelial Cells. Sci. Rep. 2016, 6, 29240. [Google Scholar] [CrossRef] [PubMed]
  165. Stec, D.E.; Hosick, P.A.; Granger, J.P. Bilirubin, Renal Hemodynamics, and Blood Pressure. Front. Pharmacol. 2012, 3, 18. [Google Scholar] [CrossRef] [PubMed]
  166. DiNicolantonio, J.J.; McCarty, M.F.; O’Keefe, J.H. Antioxidant Bilirubin Works in Multiple Ways to Reduce Risk for Obesity and Its Health Complications. Open Heart 2018, 5, e000914. [Google Scholar] [CrossRef]
  167. Nam, J.; Lee, Y.; Yang, Y.; Jeong, S.; Kim, W.; Yoo, J.-W.; Moon, J.-O.; Lee, C.; Chung, H.Y.; Kim, M.-S.; et al. Is It Worth Expending Energy to Convert Biliverdin into Bilirubin? Free Radic. Biol. Med. 2018, 124, 232–240. [Google Scholar] [CrossRef]
  168. Nobles, C.L.; Green, S.I.; Maresso, A.W. A Product of Heme Catabolism Modulates Bacterial Function and Survival. PLoS Pathog. 2013, 9, e1003507. [Google Scholar] [CrossRef] [PubMed]
  169. Vítek, L. Bilirubin as a Signaling Molecule. Med. Res. Rev. 2020, 40, 1335–1351. [Google Scholar] [CrossRef] [PubMed]
  170. Creeden, J.F.; Gordon, D.M.; Stec, D.E.; Hinds, T.D. Bilirubin as a Metabolic Hormone: The Physiological Relevance of Low Levels. Am. J. Physiol.-Endocrinol. Metab. 2021, 320, E191–E207. [Google Scholar] [CrossRef]
  171. Greaves, R.F.; Kricka, L.; Gruson, D.; Martin, H.; Ferrari, M.; Bernardini, S. Emerging Technology: A Definition for Laboratory Medicine. Clin. Chem. Lab. Med. (CCLM) 2023, 61, 33–36. [Google Scholar] [CrossRef] [PubMed]
  172. Westenberg, L.E.H.; Been, J.V.; Willemsen, S.P.; Vis, J.Y.; Tintu, A.N.; Bramer, W.M.; Dijk, P.H.; Steegers, E.A.P.; Reiss, I.K.M.; Hulzebos, C.V. Diagnostic Accuracy of Portable, Handheld Point-of-Care Tests vs Laboratory-Based Bilirubin Quantification in Neonates. JAMA Pediatr. 2023, 177, 479. [Google Scholar] [CrossRef] [PubMed]
  173. Hazarika, C.J.; Borah, A.; Gogoi, P.; Ramchiary, S.S.; Daurai, B.; Gogoi, M.; Saikia, M.J. Development of Non-Invasive Biosensors for Neonatal Jaundice Detection: A Review. Biosensors 2024, 14, 254. [Google Scholar] [CrossRef] [PubMed]
  174. Bandiera, A.; Passamonti, S.; Dolci, L.S.; Focarete, M.L. Composite of Elastin-Based Matrix and Electrospun Poly(L-Lactic Acid) Fibers: A Potential Smart Drug Delivery System. Front. Bioeng. Biotechnol. 2018, 6, 127. [Google Scholar] [CrossRef]
  175. Bergonzi, C.; d’Ayala, G.G.; Elviri, L.; Laurienzo, P.; Bandiera, A.; Catanzano, O. Alginate/Human Elastin-like Polypeptide Composite Films with Antioxidant Properties for Potential Wound Healing Application. Int. J. Biol. Macromol. 2020, 164, 586–596. [Google Scholar] [CrossRef] [PubMed]
  176. Bandiera, A. Transglutaminase-Catalyzed Preparation of Human Elastin-like Polypeptide-Based Three-Dimensional Matrices for Cell Encapsulation. Enzyme Microb. Technol. 2011, 49, 347–352. [Google Scholar] [CrossRef] [PubMed]
  177. Colomina-Alfaro, L.; Marchesan, S.; Stamboulis, A.; Bandiera, A. Smart Tools for Antimicrobial Peptides Expression and Application: The Elastic Perspective. Biotechnol. Bioeng. 2023, 120, 323–332. [Google Scholar] [CrossRef] [PubMed]
Molecules 30 00439 g001
Figure 1. The technological development of the HELP-UnaG fusion protein HUG.
Figure 1. The technological development of the HELP-UnaG fusion protein HUG.
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Figure 2. (A) Primary structure of the recombinant eight-block human elastin-like polypeptide. The gray box indicates the possible point of in-frame insertion of any domain. (B) Schematic representation of the RDL-like cloning strategy followed to obtain the multimerization of the human elastin-derived module. The first module was cloned between two different unique restriction sites of the plasmid. The cross-linking and elastin-like hydrophobic domains are highlighted in green and blue, respectively.
Figure 2. (A) Primary structure of the recombinant eight-block human elastin-like polypeptide. The gray box indicates the possible point of in-frame insertion of any domain. (B) Schematic representation of the RDL-like cloning strategy followed to obtain the multimerization of the human elastin-derived module. The first module was cloned between two different unique restriction sites of the plasmid. The cross-linking and elastin-like hydrophobic domains are highlighted in green and blue, respectively.
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Figure 3. Primary structure of the chimeric HUG polypeptide. In bold is reported the amino acid sequence of UnaG. Theoretical molecular weight is 60,406 Da.
Figure 3. Primary structure of the chimeric HUG polypeptide. In bold is reported the amino acid sequence of UnaG. Theoretical molecular weight is 60,406 Da.
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Molecules 30 00439 g004
Figure 4. Sequential steps of the cycle for purification of HUG.
Figure 4. Sequential steps of the cycle for purification of HUG.
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Figure 5. Model of the tertiary structure of HUG. The model is an AlfaFold2 minimized structure of HUG, with the UnaG domain (red ribbon) connected to the HELP biopolymer (blue ribbon).
Figure 5. Model of the tertiary structure of HUG. The model is an AlfaFold2 minimized structure of HUG, with the UnaG domain (red ribbon) connected to the HELP biopolymer (blue ribbon).
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Molecules 30 00439 g006
Figure 6. Workflow of the HUG assay. The 96-well plate is filled with standard solutions and samples (grey scale colors). After 2 h, steady-state green fluorescence (green scale colors) is recorded to calculate bilirubin concentration.
Figure 6. Workflow of the HUG assay. The 96-well plate is filled with standard solutions and samples (grey scale colors). After 2 h, steady-state green fluorescence (green scale colors) is recorded to calculate bilirubin concentration.
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Figure 7. Progression through the technology readiness levels of HUG and its use in the fluorometric assay of bilirubin, biliverdin and bilirubin glucuronide. TRL 4 was achieved by producing the new HELP-UnaG fusion protein, retaining the main functions of its individual protein domains [1]. TRL 5 was achieved by characterizing the components and processes of the HUG assay: 1. The structure and function of HUG [45], 2. The HUG purification protocol [124], 3. The preparation of bilirubin standard solutions [125], 4. The HUG assay of bilirubin in human plasma [126], and the combined analysis of bilirubin and biliverdin [127]. TRL 6 was achieved by applying the HUG assay to quantify bilirubin and/or bilirubin glucuronide and/or biliverdin in biological samples, such as: 1. The effluent of the isolated perfused rat liver [128], 2. Metastatic cancer cells grown in vitro [132], 3. The plasma collected from farmed fish or biliverdin reductase -/- C57Bl/6 mice [127], 4. Human plasma biobanks and rat blood (work in progress).
Figure 7. Progression through the technology readiness levels of HUG and its use in the fluorometric assay of bilirubin, biliverdin and bilirubin glucuronide. TRL 4 was achieved by producing the new HELP-UnaG fusion protein, retaining the main functions of its individual protein domains [1]. TRL 5 was achieved by characterizing the components and processes of the HUG assay: 1. The structure and function of HUG [45], 2. The HUG purification protocol [124], 3. The preparation of bilirubin standard solutions [125], 4. The HUG assay of bilirubin in human plasma [126], and the combined analysis of bilirubin and biliverdin [127]. TRL 6 was achieved by applying the HUG assay to quantify bilirubin and/or bilirubin glucuronide and/or biliverdin in biological samples, such as: 1. The effluent of the isolated perfused rat liver [128], 2. Metastatic cancer cells grown in vitro [132], 3. The plasma collected from farmed fish or biliverdin reductase -/- C57Bl/6 mice [127], 4. Human plasma biobanks and rat blood (work in progress).
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Figure 8. Spectrum of applicability of the HUG assay in biology and medicine.
Figure 8. Spectrum of applicability of the HUG assay in biology and medicine.
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Figure 9. Biosynthesis of bile pigments. Heme is cleaved in a NADPH+H+-dependent reaction catalyzed by heme oxygenase, whose products are biliverdin, CO and Fe2+. Bilirubin is the product of the reaction catalyzed by NAD(P)H+H+-dependent biliverdin reductase. The reaction scheme is in accord with Ref. [135].
Figure 9. Biosynthesis of bile pigments. Heme is cleaved in a NADPH+H+-dependent reaction catalyzed by heme oxygenase, whose products are biliverdin, CO and Fe2+. Bilirubin is the product of the reaction catalyzed by NAD(P)H+H+-dependent biliverdin reductase. The reaction scheme is in accord with Ref. [135].
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Table 1. Optimized conditions for HELP expression in E. coli [27].
Table 1. Optimized conditions for HELP expression in E. coli [27].
ConditionSpecification
Medium and SuppleTB, terrific broth
PGB, Phosphate buffer with glycerol
Recombinant SystemNEBExpress®Iq Competent E. coli (High Efficiency)
Induction OD6000.9–1
IPTG final concentration0.1 mM
Growth after induction4 h
Recombinant protein yield180 mg/L
Table 2. Inverse transition temperatures of HELP at different solution conditions obtained by turbidimetric measurements.
Table 2. Inverse transition temperatures of HELP at different solution conditions obtained by turbidimetric measurements.
C
mg/mL		SolutionTt
°C
2.0PBS, pH = 7.433
2.0Tris, pH = 8.0
Tris, pH = 8.0, 0.15 M NaCl		30
36
2.0NaPi, pH = 7.3
NaPi, pH = 7.3, 0.15 M NaCl		30
39
Table 3. Differential scanning calorimetry results of HELP solutions in different solvent conditions.
Table 3. Differential scanning calorimetry results of HELP solutions in different solvent conditions.
SolventC
mg/mL		Tt
°C		ΔHtr
kJ/mol		ΔStr
J/molK
PBS pH = 7.443421623.9
10 mM Tris pH = 8
10 mM Tris pH = 8, 0.15 M NaCl		8
8		29
34		198
35		655
114
10 mM NaPi pH = 6.8
10 mM NaPi pH = 6.8, 0.15 M NaCl		8
8		29.5
39.5 		6.67
0.141		21.8
0.45
Table 4. Synopsis of uses of UnaG in experimental biology and medicine.
Table 4. Synopsis of uses of UnaG in experimental biology and medicine.
ApplicationPurification MethodReferences
Bile pigment detection in biological fluid, cells and medium-[67,70,71,72,73,74,75]
Ni2+ or glutathione-affinity chromatography[76]
Ni-NTA Fast Start kit or similar[77,78,79]
Live-cell imagingAnti-FLAG immuno-purification from cell lysate[80]
Ni2+-NTA agarose beads[81]
-[77,82,83,84,85,86,87,88]
Lumirubin detection in urineNi2+ or glutathione-affinity chromatography [3,89,90]
Live-cell imaging in bacteriaMBP-trap and His-trap[91,92,93,94,95]
Bilirubin in tissuesCrude bacterial lysate (no further purification)[96,97]
Probe for yeast display-[98]
BVR assayNi2+-NTA Fast Start kit[79]
As gene reporter -[99,100,101,102,103,104,105,106,107,108]
Probe for anoxic/hypoxic conditions-[69,109,110,111,112,113,114,115,116,117,118,119]
Fluorescent probe with reversible switching in cells and tissuesGST-purification; ion exchange Chr; size-exclusion chr[120]
Structural and functional studiesNi2+ or glutathione-affinity chromatography[121]
MBP-trap and His-trap; gel filtration[84]
His-trap and desalted with a PD-10 column [66]
-[122,123]
Table 5. Validation parameters of the HUG assay in the range 0.05–50 nM using standard bilirubin solutions containing 0.4 g/L BSA.
Table 5. Validation parameters of the HUG assay in the range 0.05–50 nM using standard bilirubin solutions containing 0.4 g/L BSA.
Validation ParameterValue
Linearityrange0–75 nM
slope785
R20.9990
LOD0.05–10 nM0.36 nM
0.5–50 nM1.56 nM
LOQ0.05–10 nM1.10 nM
0.5–50 nM4.75 nM
Accuracy (relative error)range1–9%
median4.5%
Precision with standard solutions
(coefficient of variation)		range1.7–5.8%
median2.6%
Precision with human plasma
(coefficient of variation)		range1.9–12.6%
mean6.7%
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MDPI and ACS Style

Sist, P.; Urbani, R.; Tramer, F.; Bandiera, A.; Passamonti, S. The HELP-UnaG Fusion Protein as a Bilirubin Biosensor: From Theory to Mature Technological Development. Molecules 2025, 30, 439. https://doi.org/10.3390/molecules30030439

AMA Style

Sist P, Urbani R, Tramer F, Bandiera A, Passamonti S. The HELP-UnaG Fusion Protein as a Bilirubin Biosensor: From Theory to Mature Technological Development. Molecules. 2025; 30(3):439. https://doi.org/10.3390/molecules30030439

Chicago/Turabian Style

Sist, Paola, Ranieri Urbani, Federica Tramer, Antonella Bandiera, and Sabina Passamonti. 2025. "The HELP-UnaG Fusion Protein as a Bilirubin Biosensor: From Theory to Mature Technological Development" Molecules 30, no. 3: 439. https://doi.org/10.3390/molecules30030439

APA Style

Sist, P., Urbani, R., Tramer, F., Bandiera, A., & Passamonti, S. (2025). The HELP-UnaG Fusion Protein as a Bilirubin Biosensor: From Theory to Mature Technological Development. Molecules, 30(3), 439. https://doi.org/10.3390/molecules30030439

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