Biotechnology Advances 54 (2022) 107817 Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Research review paper Insights on the emerging biotechnology of histidine-rich peptides Hèctor López-Laguna a, b, c, 1, Eric Voltà-Durán a, b, c, 1, Eloi Parladé a, b, c, 1, Antonio Villaverde a, b, c, *, Esther Vázquez a, b, c, *, Ugutz Unzueta b, c, d, * a Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain b Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain c CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain d Biomedical Research Institute Sant Pau (IIB Sant Pau), Sant Antoni Mª Claret 167, 08025 Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: In the late 70’s, the discovery of the restriction enzymes made possible the biological production of functional Recombinant protein proteins by recombinant DNA technologies, a fact that largely empowered both biotechnological and pharma­ Modular protein ceutical industries. Short peptides or small protein domains, with specific molecular affinities, were developed as Biomaterials purification tags in downstream processes to separate the target protein from the culture media or cell debris, Biosensing Nanobiotechnology upon breaking the producing cells. Among these tags, and by exploiting the interactivity of the imidazole ring of histidine residues, the hexahistidine peptide (H6) became a gold standard. Although initially used almost exclusively in protein production, H6 and related His-rich peptides are progressively proving a broad applica­ bility in novel utilities including enzymatic processes, advanced drug delivery systems and diagnosis, through a so far unsuspected adaptation of their binding capabilities. In this context, the coordination of histidine residues and metals confers intriguing functionalities to His-rich sequences useable in the forward-thinking design of protein-based nano- and micro-materials and devices, through strategies that are comprehensively presented here. 1. Introduction Since the 70’s (Porath et al., 1975) and along decades (Gaberc-Por­ ekar and Menart, 2001; Porath, 1992; Riguero et al., 2020; Spries­ Protein purification is an essential practice in biotechnology, drug tersbach et al., 2015; Sulkowski, 1985), immobilized metal affinity development and proteomics, and a late downstream step in the re­ chromatography (IMAC) has proved to be a robust choice method for combinant protein production task flow (Garg et al., 1991; Gräslund preparative protein purification because of its versatility, cost effec­ et al., 2008). Considering the diversity of cell factories and target pro­ tiveness, scalability and simplicity, compliant with high-throughput teins in the protein production scenario, simple, one-step separation requirements. The IMAC concept is supported by the relatively strong protocols are ideally required to fulfill the demands for generality, interactions between transition metal ions (namely Ni2+, Cu2+, Zn2+ and scalability, and high throughput (Fig. 1A). In this context, affinity tags, Co2+) and the side chains of some particular amino acids. Among them, namely peptides or proteins with selectivity for specific binders have the amino acid histidine is the tightest binder through the electron donor been incorporated to recombinant proteins by genetic fusion, usually at group on its imidazole ring, that forms coordination bonds with the either the amino or carboxy termini, to selectively retain the whole metal (Watly et al., 2014). If immobilized in a matrix, the metal acts as a fusion in affinity chromatography systems based on immobilized ligands molecular hook for histidine-rich protein segments, that can be further (Amarasinghe and Jin, 2015; Kimple and Sondek, 2004; Mishra, 2020). eluted from the matrix by adjusting the buffer pH or by adding excess Essentially, any pair of interactors involving at least a peptidic partner imidazole. Since series of consecutive histidine residues confer a much should be suitable for the design of entrapment protocols to retain a tighter binding than isolate amino acids (Bornhorst and Falke, 2000; specific protein among complex molecular mixtures such as cell lysates Gaberc-Porekar and Menart, 2005), polyhistidine stretches and specially or culture media. H6, have become gold standard affinity tags for protein purification * Corresponding authors. E-mail addresses: antoni.villaverde@uab.cat (A. Villaverde), esther.vazquez@uab.cat (E. Vázquez), uunzueta@santpau.cat (U. Unzueta). 1 Equally contributed. https://doi.org/10.1016/j.biotechadv.2021.107817 Received 10 April 2021; Received in revised form 16 July 2021; Accepted 16 August 2021 Available online 19 August 2021 0734-9750/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). H. López-Laguna et al. Biotechnology Advances 54 (2022) 107817 (Table 1). In this sense, smaller tags with three to five histidine residues purification column,in series, to catch leaked ions). An alternative so­ can also serve for this purpose (López-Laguna et al., 2019) at the expense lution is to eliminate eluted ions during the protein dialysis step using a of a weaker binding. On the other hand, longer histidine tags (e.g. H8 or chelator such as EDTA, although this chemical compound can also be a H10) are also an alternative when high purity is needed and optimiza­ contaminant for the protein of interest and it must be subsequently tion of H6-tags is not sufficient (Grisshammer and Tucker, 1997). removed (Mónico et al., 2017). Several downsides, however, include the possibility of inhibiting protein Advanced strategies based on this principle have been developed to functions, inducing conformational changes, decreasing the protein purify His-tagged proteins in a more efficient, sustainable, easier or expression, or requiring higher imidazole concentrations to elute, which cheaper manner. In this regard, magnetic nanoparticles have emerged as ultimately lead to unstable proteins in the solution (Mohanty and a simple, fast, and high-throughput system to purify proteins from Wiener, 2004). Nevertheless, it is historically assumed that H6 is an complex bacterial or animal cell lysates by magnetic separation when appropriate choice when designing recombinant proteins unless previ­ functionalized with linker-chelating metal ions (Xu et al., 2004), nickel ous concerns regarding the desired number of histidine residues exist. silicate (Wang et al., 2014a) or simply a nickel coating (Minkner et al., The binding mediated by histidine residues occurs since at physiological 2020). In 2019, Schwaminger and collaborators went a step further with pH, the molecular coordinative foci of imidazole rings are found in the a proof of principle of a magnetic one-step purification system, using δ1-Nitrogen atom, which is capable to act as a nucleophilic center and non-functionalized bare iron oxide nanoparticles (BIONs) (Schwaminger share two unpaired electrons with the desired targeting molecule, which et al., 2019). Besides IMAC, liquid-liquid extraction with affinity ionic will act as an electrophilic Lewis acid. In particular, the d-orbitals of liquids has been also used for the same purpose, resulting in a suitable these electrophiles allow the formation of coordinative bonds whose method for large-scale purification (Ren et al., 2015). His-tagging has geometrical structures promote the simultaneous gluing of several served for the purification of structures more complex than single imidazole rings from adjacent His residues (Valenti et al., 2006). This polypeptides, such as bacterial outer membrane vesicles (OMVs) (Alves type of interaction is strongly held into the structure formed by the et al., 2017), virus-like particles (Gupta et al., 2020) or bacteriophages molecular partners in the complex, supporting mechanical forces that (Ceglarek et al., 2013). sustain the applicability of polyhistidine tails as molecular ligands An additional step in the enhancement of IMAC purification exploits (Lopez-Laguna et al., 2019). nitrilotriacetic (NTA)-Ni2+-His tag coordination complexes (Fig. 1B). NTA is an aminopolycarboxylic acid with a tripodal tetradentate nature, 2. Advanced systems for protein purification able to form coordinative interactions with divalent metal ions in a bonding type called chelation. The NTA-metal ion complexes can spe­ The traditional IMAC, with divalent cations immobilized on agarose cifically interact with certain chemically compatible amino acid resi­ resin beads, basically Sepharose, remains as a conventional protocol dues. Among all different options, imidazole rings from histidine (Loughran et al., 2017). Here, several divalent cations (Co2+, Cu2+, residues are extremely good targets, as they offer two non-bonded va­ Zn2+, Ni2+) are useful for purification purposes and each of them shows lences on their surface that are available for further metal ion coordi­ different binding affinity towards histidine. Its choice therefore, depends nation. In a general manner, more-than-two NTA (bis or tris) structures on the final application. Co2+ for example, forms less stable coordination are needed to ensure a sufficient strong binding between ion and histi­ bonds with histidine residues and is especially suitable when high pro­ dine (Fig. 1B). As a result, ternary bis or tris NTA-metal ion-histidine tein purity is required. On the other hand, Cu2+ has the greatest affinity stable complexes are formed. From here, the chemical versatility of NTA and is useful for enriching low abundant proteins from crude lysates, allows this technology to be used in a plethora of different protein en­ prior to other purification steps. Ni2+ and Zn2+ have an intermediate gineering fields (Wijeratne et al., 2016). affinity, being nickel the most extended choice (Riguero et al., 2020). In Although poorly studied in general terms, His-rich tags are suspected all these cases, divalent cations might suffer from leakage from IMAC to have potential undesired impacts on the structure, function and columns and elute with the protein of interest. For some particular ap­ immunogenicity of some of the target proteins (Khan et al., 2012; Singh plications, the presence of metal ions may affect protein activity or et al., 2020). Such side-effects of His-tagging, mild and case-dependent, promote undesired reactions. Therefore, in these cases, we must ensure can be solved by the proteolytic removal of the peptide upon purifica­ that the final product is free of divalent cations. In this sense, some tion, driven by a protease target site introduced during the genetic commercial protein purification companies have already developed fusion (Goh et al., 2017) or by regulatable, Ni2+-mediated hydrolysis resins that show tighter bonds with the divalent cation and propose (Abd Elhameed et al., 2019). More details about the potential bottle­ strategies to diminish its presence in the eluted fractions (e.g. using necks generated by His-tagging are discussed below. Because of these prepacked columns, pre-washing the column with elution buffer prior to issues, alternative affinity tags have been extensively used, such as small protein purification or connecting an uncharged column just after the peptides (FLAG, c-Myc, Strep-Tag) or longer polypeptidic ligands such Fig. 1. A. Schematic process to obtain a pure re­ combinant polypeptide (e.g. His-tagged Human Serum Albumin, PDB: 1AO6) by means of a His-tag genetically fused to the target protein. The sequence of activities involves gene design (in which the desired protein-encoding gene is fused to the selected tag), cloning, transformation in a capable cell factory, production in an adequate scale (lab, pilot or indus­ trial), His-tag based immobilization in an IMAC col­ umn (I) and subsequent displacement using imidazole (II). B. Zoomed representation of the coordination bonding between Ni-NTA and the histidine imidazole ring. 2 H. López-Laguna et al. Biotechnology Advances 54 (2022) 107817 Table 1 A summary of applications of His-rich tags in protein sciences. Application Strategy Used material or technical basis Approaches Representative references category Protein Traditional Immobilized Metal Ion Affinity Sepharose beads with immobilized divalent Loughran et al. (2017) purification Chromatography Chromatography (IMAC) metal ions Alternative Material- Magnetic NPs - Linker chelated metal ion NPs. Minkner et al. (2020); Schwaminger et al. based strategies - Nickel (II) Silicate NPs. (2019); Wang et al. (2014a); Xu et al. - Nickel (II) Coated NPs. (2004) - Iron oxide NPs (BIONs). Alternative Liquid-based Affinity ionic liquid (AIL) Liquid-liquid extraction with affinity ionic Ren et al. (2015) strategies liquids Protein labelling Traditional Ab-protein Western Blot Anti His-tag Abs emitting detectable signals Hirano (2012); Kreisig et al. (2014) and detection recognition 99m His-tag radiolabeling Tc(CO)3-His-tag Technetium-99 attached to His-tags, emitting Garousi et al. (2020) radioactive signals scFv-Bioimaging Histidine tagged monoclonal scFv Abs recognizing biological compounds Min and Yamabhai (2020) scFv and His-rich tags allowing cell or tissue imaging (e.g. by immunofluorescence, immunoelectron microscopy, ELISA or Western Blot) Mono or Tris NTA-based Signaling compound-NTA-Ni2+- NTA- Ni2+ complexes label His-tagged proteins Banerjee et al. (2016); Chao et al. (2017); complexes His-tag and conjugated compounds (e.g. ATTOX, Kitai et al. (2011); Kollmannsperger et al. ALEXAX, OG488, FEW646, BODIPY, arylazide, (2016); Soh (2008); Wang et al. (2014b); QD and gold nanoparticles) emit detectable Zheng et al. (2016) signals Protein Basic Immobilization Surface-NTA-Ni2+-His tagged NTA-Ni2+ complexes conjugated to chemically Dai et al. (2018); Kang et al. (2017); Liu immobilization strategies for biosensing proteins adequate surfaces allowing the detection of et al. (2010); Mikula et al. (2018); His-tagged proteins, nucleic acids, biomarkers, Povedano et al. (2020); Ravikumar et al. toxins, immunoglobulins or pesticides (2018); Vallina-Garcia et al. (2007); Zhang et al. (2014) Alternative Au-coated probes-NTA-Co3+-His- Au-coated probes conjugated with NTA-Co3+ Horta et al. (2020) Immobilization tagged proteins complexes used to create SPR Optic biosensors strategies for biosensing immobilizing His-tagged antigens Chitosan-Zn2+-His-tagged Chitosan-Zn2+ complex immobilize and allow Soleri et al. (2015) proteins in a quartz crystal the detection of His tagged mycotoxin in microbalance (QCM) hapten biosensors using the QCM technology Biocatalysis in glass Fe3+ porosity glass carriers with Controlled porosity Fe3+ glass carriers used to Pellis et al. (2017) surfaces immobilized His-tagged enzymes immobilize His-tagged enzymes to catalyze reactions Biocatalysis in self- Zn2+ assisted generation of Generation of H6-tagged β-galactosidase Sanchez et al. (2021) immobilized insoluble reusable insoluble protein microscale granules with enhanced stability enzymes microgranules Biocatalysis in Semicontinuous or continuous His-tagged enzymes immobilized in different Britton et al. (2017); Plz et al. (2020) bioreactors -flow bioreactors with His-tag functioning bioreactors promoting successive immobilized enzymes and multi-step product transformations Microfluidic bioreactors with Microfluidic packed-bed bioreactors using Peschke et al. (2019) magnetic beads attaching His- magnetic beads to attach His-tagged enzymes tagged enzymes to perform desired enzymatic reactions Bioengineering Glass-plate Ni2+ chelated surface Glass-plate Ni2+ surfaces used to enhance in Shakya et al. (2020) with immobilized His-tagged vitro proliferation of human mesenchymal cells growth factor by immobilizing FGF Protein site- Site specific conjugation Desired compound-NTA-Ni2+- Biological compounds or materials such as Goodman et al. (2009); Rai et al. (2016); specific by metal cation- His-tagged proteins DNA, nanolipoproteins (MPLA-NiNLP), Au- Shimada et al. (2008); Wilkins et al. conjugation coordination. polymer NPs or QD can be chemically attached (2019); Zheng et al. (2016) to NTA-Ni2+ complexes and be specifically coordinated to His-rich tags from proteins Site specific conjugation Baylis-Hillman ester - ternary Ternary NTA-Ni2+ complexes can be used to Liu and Melman, 2013) by alkynyl group NTA-Ni2+ bring an alkynyl group-carrying ester moiety addition near the imidazole ring of a desired histidines for its two stage derivatization with molecules. Site specific conjugation Cation free Sulfone – His bis- Sulfone-mediated bis-alkylation of PEG-bis- Cong et al. (2012); Peciak et al. (2019) by histidines bis- alkylation. sulfone or PEG10-mono-sulfone molecules to alkylation His-tag in desired proteins (e.g. interferon α-2a or anti-TNFα domain Ab) Site specific conjugation Michael addition of 2-cyclohexa­ Orthogonal late-stage installation of aminooxy Joshi and Rai (2019) by cyclic ketone none in His residue. functionalized NMR tags or dyes in exposed generation His residues on protein surface (e.g. lysozyme C) Site specific conjugation D-gluconic acid δ-lactone (GDL) Site specific acylation allows the direct Martos-Maldonado et al. (2018) by histidine acylation. and 4-methoxyphenil esters for addition of a biotin or an azide functional specific acylation of amino- group for its subsequent reaction with DBCO- terminal His-tags (GHHHn). functionalized molecules into His-rich tags of different proteins (e.g. GFP, MBP or SUMO). Protein Ion-based protein gluing His-tag/divalent cation/His-tag Divalent cations used as coordinative agents of Bai et al. (2013); Brodin et al. (2015); assembling to manufacture soluble His-tags (interacting with the δ1 nitrogen of Cespedes et al. (2018); Dexter et al. (2006); nanostructures the histidine imidazole ring) and promoting Diaz et al. (2018); Haglin et al. (2017); (continued on next page) 3 H. López-Laguna et al. Biotechnology Advances 54 (2022) 107817 Table 1 (continued ) Application Strategy Used material or technical basis Approaches Representative references category adequate nanostructuring (10-350 nm, e.g. Maniaci et al. (2019); Serna et al. (2020a); dimers, coiled coils, nanodiscs, nanocapsules, Serna et al. (2020c); Tunn et al. (2018); nanorings, helical nanotubes, nanowires, Unzueta et al. (2020); van Eldijk et al. interfacial films or tumor targeted NPs) in a (2016); Zhang et al. (2012) reversible way (upon addition of EDTA) Ion-based protein gluing His-tag/divalent cation/His-tag Divalent cations used as precipitator agents to Chen et al. (2019); Jiang et al. (2011); to manufacture create functional secretory-like granules at the Sanchez et al. (2020); Serna et al. (2020b) insoluble functional microscale in a reversible way (upon addition depots of EDTA) Ab: Antibody; scFv: Single-chain variable fragments; Ni2+: Nickel (II); NTA: Nitrilotriacetic acid; ATTOX & ALEXAX: ATTO565 or ATTO655 and ALEXA647 or ALEXA488; QD: Quantum Dots; SPR: Surface plasmon resonance; Au: Gold; Co3+: Cobalt (III); FGF: Fibroblast growth factor; DTPC: DNA-template protein conjugation strategy; PEG: Polyethylene Glycol; NMR: Nuclear magnetic resonance; GFP: Green fluorescent protein; MBP: Maltose-binding protein; SUMO: Small ubiquitin-like modifier protein; EDTA: Ethylenediaminetetraacetic acid; DBCO: Dibenzocyclooctyne. as Maltose Binding Protein (MBP) or Glutathione S-Transferase (GST) virus (Zheng et al., 2016), or Au NPs NTA-Ni2+ labelling dynactin sub­ (Pina et al., 2014). Also, several novel tags are already under develop­ unit p62 (Kitai et al., 2011)), protein function (e.g. labelling the DNA ment (Spy Tag, Strach Binding Protein, Fluropatite, Diatomite, Beta- repair protein XPA122 (Chao et al., 2017)), subcellular location (e.g. GRP) trying to reduce protein contaminants and/or production costs tracing extracellular and intracellular histidine-tagged proteins (Wang (Khairil Anuar et al., 2019; Wood, 2014). Related to that, a specific et al., 2014b), protein targeting, biodistribution and general protein solution to increase His-tagged protein purity is the use of low back­ pharmacokinetics (Garousi et al., 2020). The tripodal tetradentate na­ ground cell factory strains, such as Escherichia coli LOBSTR, which is ture of NTA allows the eventual interaction with exposed H6 tails engineered to eliminate major His-rich contaminants (Andersen et al., (Raghunath and Dyer, 2019). Besides the binding affinity of mono-NTA 2013). is sufficient for IMAC needs (KD: 10 × 10-6 M), the molecular labelling of polyhistidine-tagged proteins requires tighter associations to form stable 3. Protein labelling and detection complexes useful in biological conditions. Because of that, three NTAs are usually associated, named Tris-NTA, to provide sufficient binding 3.1. Protein immunodetection affinity (KD: 20 × 10− 9 M (Huang et al., 2006)) and to increase the complex stability up to 4 orders of magnitude, thanks to the multi­ Specific protein recognition appears as a prior method in proteomic valency of the system. Tris-NTA-Ni2+ coordinated complexes are being profiling, and monoclonal or polyclonal antibody (Ab)-protein immu­ used by the scientific community to interact with inner-cell histidine nodetection has raised as one of the most popular methodologies for tagged proteins without affecting endogenous processes, presenting protein identification (Signore et al., 2017). Upon Western Blot, protein high specificity and low cell toxicity (Kollmannsperger et al., 2016). detection ensures adequate polypeptide identification and quantifica­ These complexes can be bound to H6-tagged cell penetrating peptides tion even in complex samples (Obermaier et al., 2015). Considering the (CPPs) to promote cell entrance, and afterwards, spontaneously change large number of H6-tagged recombinant proteins (Loughran et al., its preference to bind H10-tagged proteins of interest inside the cell, due 2017), the importance of using anti-polyhistidine tag antibodies has to the 10-fold higher binding affinity of the longer tag (Schneider and emerged in parallel (Kreisig et al., 2014). In anti-H6-based detection Hackenberger, 2017). Also, NTA-Ni2+-red fluorescent probes or similar techniques (Table 1), a primary monoclonal Ab recognizes the H6 tail, (e.g. AB or BODIPY) complexes can enter into bacteria cells to selec­ followed by a secondary Ab that recognizes the first attached Ab tively label histidine-tagged proteins by using appropriate amounts of (Fig. 2A). Primary (or secondary, binding to primary) Abs are typically detergents (e.g. Tween 80 (Chao et al., 2017)). The traditional proteo­ labelled with organic fluorophores (e.g. Alexa Fluor) or enzymes (e.g. mic profiling techniques, together with protein radio-labelling methods alkaline phosphatase and peroxidase) that emit a measurable signal and NTA-Ni2+-based complexes (Fig. 2B), outpoint the huge versatility (Hirano, 2012) allowing its quantitative detection. and applicability of using H6 (or related tags) as labelling target for protein characterization and in situ functional imaging. 3.2. Tracking oriented protein labelling 4. Protein immobilization in biosensing, biocatalysis and bioengineering Interestingly, the popularity of using NTA-Ni2+-His tag coordination complexes in protein purification has uplifted in novel protein engi­ Protein immobilization onto solid surfaces has several applications neering fields (Johnson and Martin, 2005) and single molecule imaging, in protein-based biocatalysis, biosensing and bioengineering (Table 1). along with recent H6 radiolabeling (e.g. 99mTc(CO)3) (Garousi et al., In this sense, among the growing variety of developed protein immo­ 2020) and single-chain variable fragments (scFv) of monoclonal Abs bilization methods, including entrapment, adsorption, cross-linking or bioimaging (Min and Yamabhai, 2020). Novel formulations of NTA- covalent binding, affinity techniques provide a highly specific, based complexes are being designed to ensure precise protein detec­ controllable, and reversible way to anchor proteins to surfaces without tion at sub-nanomolar concentrations and to create clear pictures of losing their functional structure (Ley et al., 2011; Zhang et al., 2014). protein spatiotemporal behavior under physiological conditions (Koll­ Additional benefits of this approach are protein stabilization, protection mannsperger et al., 2016). The specific conjugation of chromophores (e. against environmental stresses and reusability in successive processes g. ATTO565, ATTO655, ALEXA647, ALEXA488, OG488, and FEW646 (Koll­ (Rodrigues et al., 2013). Taking advantage of their affinity towards mannsperger et al., 2016; Soh, 2008)), small molecules (e.g. distylryl divalent metal cations, surfaces or nanomaterials can be decorated with BODIPY or arylazide crosslinkers (Chao et al., 2017)) and other nano­ His-tagged proteins, rendering uniformly oriented protein layers with scale materials (e.g. quantum dots, QD (Banerjee et al., 2016) or gold controlled spatial distribution and without multipoint attachments that nanoparticles (Kitai et al., 2011)) to these complexes allows exploring could impair their functionality (Ley et al., 2011). This chemistry works biological fundamental processes such as viral infections, protein dy­ well under mild conditions, at physiological pH and in presence of salts, namics and mobility (e.g. QD-NTA-Ni2+ labelling respiratory syncytial 4 H. López-Laguna et al. Biotechnology Advances 54 (2022) 107817 Fig. 2. A. Overview of size, common labeling strategies, and popular detection techniques available for the study of His-tagged proteins using antibodies. B. Size comparison and structure of the Tris-NTA ligand (I), along with common labeling approaches (radiolabeling with Technetium 99m (II) or Tris-NTA-based labeling with gold nanoparticles (III), fluorescent dyes (IV) and quantum dots (V)). C. Schematic representation of direct techniques to study subcellular localization of his- tagged proteins in vitro (I), co-localization of different his-tagged proteins in vitro (II), or protein bioaccumulation in in vivo models (III). making it appropriate for applications in biological interfaces (Wegner Zhang et al., 2014) devices and also in piezoelectric transducers (Baltus et al., 2016). The conventional procedure to immobilize His-tagged et al., 2007; Li et al., 2016; Nam et al., 2013; Wegner et al., 2016). proteins involves an earlier step of surface functionalization with NTA, Optical biosensors, mainly based on Surface Plasmon Resonance (SPR) followed by the incorporation of the divalent cation, typically Ni2+ (Liu or Biolayer Interferometry (BLI), are extensively employed to detect the et al., 2010). In such process, a deep consideration of surface pretreat­ interaction of an immobilized His-tagged protein with its ligand, due to ment, metal leaching and unwanted dissociation of the His-rich tag from a change in the light properties (Fig. 3A). For example, Horta and co- the divalent cation-NTA pair is suited (Ravikumar et al., 2018). In this workers have recently developed an optical-fiber-based SPR biosensor regard, a plethora of procedures are emerging to solve these issues to detect autoantibodies in autoimmune diseases, by immobilizing His- whilst maintaining the benefits of His-tagged immobilization. For tagged antigens on Co3+-NTA Au-coated probes (Horta et al., 2020). A instance, alternative strategies involve the use of multiple NTA groups similar approach, involving Co3+-His complexes, has been used for (Lata et al., 2005), longer His-rich tags (Lata et al., 2005), different immobilizing virus-like particles in a BLI biosensor that detects nor­ chelators (e.g. chitosan (Ravikumar et al., 2018)), acbztacn (Johnson ovirus antibodies from human serum samples (Auer et al., 2017). and Martin, 2005), 1-acetato-4-benzyl-triazacyclononane (Wollenberg Moreover, the chemokine receptor CXCR4 has been successfully et al., 2014), iminodiacetatic acid (Zhao et al., 2017), NiO (Jia et al., immobilized for studying its interaction with nanobody-Fc ligands, 2016) and polydopamine (Yang et al., 2015)) or the conversion to which represents an important progress in the study of G protein- different metal oxidation states (namely Fe3+ (Pellis et al., 2017) and coupled receptors involved in a broad range of diseases (Boonen et al., Co3+ (Wegner et al., 2016)). In this last case, the process requires a first- 2020). Electrochemical biosensors tend to be sensitive, fast, simple, step of complexation between histidines and a Co2+ ion, followed by a cheap, and easy-to-miniaturize devices that detect a change in electrical subsequent Co2+ ion oxidation into Co3+, resulting in a tight immobi­ properties in the media (Dai et al., 2018; Ley et al., 2011). Their use is lized complex. specially promising in point-of-care facilities, but their susceptibility to electrochemical interferences such as pH or ionic strength changes must be faced. His-tagged proteins in electrochemical biosensors have been 4.1. Biosensing used for the detection of nucleic acids (Dai et al., 2018; Povedano et al., 2020), biomarkers (Ta et al., 2016), toxins (Vallina-Garcia et al., 2007), In biosensors, His-tagged proteins have been broadly used in optical immunoglobulins (Kang et al., 2017; Mikula et al., 2018) or pesticides (Auer et al., 2017; Avsar et al., 2020; Faccio et al., 2016; Horta et al., (Zhang et al., 2014). The quartz crystal microbalance (QCM) is a simple, 2020; Lata et al., 2005; Ravikumar et al., 2018) and electrochemical label-free, real-time, and efficient tool for the detection of biomolecular (Dai et al., 2018; Povedano et al., 2020; Vallina-Garcia et al., 2007; 5 H. López-Laguna et al. Biotechnology Advances 54 (2022) 107817 Fig. 3. A. Schematic representation of biolayer interferometry (left) and surface plasmon resonance (right) in his-tag based biosensing. B. Schematic disposition of immobilized enzymes for a sequential biocatalysis in which the product of the first reaction acts as substrate for the second reaction. Common cations used in immobilization are Ni2+, Fe3+, Co2+ or Co3+. C. His-tagged protein coating as a bioengineering strategy to enhance cell growth. D. Schematic representation of how histidine residues promote protein assembly and secretory granule-like structures with the addition of divalent cations. E. Representation of the wide range of nanoscale protein materials that can be assembled by histidine coordination with metal cations such as coiled coils (I), nanowires (II), nanorings (III), toroids (IV), nanocapsules (V) and nanotubes (VI). F. Proton sponge effect; illustration of how the decrease in pH in endosomes in presence of his-tagged proteins results in the endosomal disruption and subsequent protein liberation. interactions that is based in the measurement of the frequency decrease a molar excess of Zn2+ as mechanically stable microscale granules, a caused by a mass increase (upon interaction) in the functionalized sur­ format that allows reusability and that enhances the operational sta­ face (Nam et al., 2013). Its major drawback is the lack of sensitivity for bility of the enzyme (Sanchez et al., 2021). On the other hand, there is a small molecules. In a recent report it has been demonstrated the feasi­ growing tendency to synchronize purification and immobilization of bility of QCM to explore the binding properties of a GPCR to its immo­ His-tagged enzymes in one-step procedures, avoiding the initial on- bilized partner, which provides the basis for studying other similar column step and directly working with complex protein mixtures interactions through this technique (Avsar et al., 2020). Finally, myco­ derived from cell lysates (Wang et al., 2017). Britton and collaborators toxin recognition peptides have been also immobilized over Zn2+ developed a continuous-flow bioreactor with a 10-min synchronized adsorbed chitosan foam via His-rich tag for the development of hapten purification and immobilization strategy (Britton et al., 2017). More­ biosensors (Soleri et al., 2015). over, they were able to produce a multi-step bioreactor that allows successive transformations by controlling spatial distribution of each 4.2. Biocatalysis enzyme throughout the bioreactor surface (Fig. 3B). The same principle has been applied in semi-continuous bioreactors (Plz et al., 2020). In biocatalysis, immobilization enhances enzymatic stability and Magnetic nanoparticles have been widely used for such dual purpose for facilitates product recovery and enzyme reusability. His-rich tag-based both intracellular and extracellular proteins obtaining great enzyme immobilization has been used for many different reactions in different reusability indexes after several batches (Yang et al., 2015; Zhao et al., supports. For example, EnginZyme® has developed Fe3+-chelated glass 2017; Zhou et al., 2017). Peschke and co-workers have exploited the use carriers with controlled porosity in which His-tagged enzymes can be of magnetic beads in a microfluidic packed-bed bioreactor with His- successfully immobilized and perform their activities (Pellis et al., tagged loaded ketoreductase (Peschke et al., 2019). The use of minia­ 2017). Also, H6-tagged β-galactosidase has been self-immobilized using turized reactors may be notably important in the future development of 6 H. López-Laguna et al. Biotechnology Advances 54 (2022) 107817 sustainable production processes. successful derivatization of recombinant proteins with a reactive alkynyl group that undergo subsequent cycloaddition of molecules (Liu and 4.3. Bioengineering Melman, 2013). As a similar approach, the DNA-template protein conjugation strat­ His-tagged protein immobilization is also useful for bioengineering egy (DTPC) allows the initial direction of an NTA-modified oligonucle­ (Fig. 3C). For example, the Fibroblast Growth Factor (FGF) was immo­ otide to a His-tagged protein. This serves then as a template for bilized onto the surface of a Ni2+ chelated glass-plate to enhance in vitro complementary N-hydroxysuccinimide (NHS)-bearing molecule conju­ proliferation of human mesenchymal stem cells (Shakya et al., 2020). gation to a lysine residue, in the close proximity of the histidine-rich tag The successful outcome can be directly attributed to the structural (Rosen et al., 2014). integrity of the FGF domain in the fusion protein. As it has been done for biosensors, Ni2+ has been replaced by Co3+ to stabilize the bond be­ 5.3. Bis-alkylation of histidine residues tween His-rich tag and NTA. Di Russo and co-workers proved that such replacement results in the development of a higher and broader range of A more sophisticated His-rich tag specific conjugation methodology E-cadherin adhesion forces, leading to extended cell spreading and relies on the bis-alkylation of two histidine residues located close colony organization (Di Russo et al., 2019). This principle is highly together in the target protein by sulfone-containing molecules, without relevant for further development of functionalized surfaces for cell the need of any metal chelator. Following this strategy, molecules such culture. as PEG have been successfully attached to different His-tagged proteins using PEG-bis-sulfone or PEG10-mono-sulfone molecules. Moreover, this 5. Site-specific conjugation type of conjugation was successfully achieved using different types of polyhistidine tags containing both continuous (H2, H6, H8) or discon­ The functionalities of proteins or protein nanomaterials can be tuned tinuous (-HGH or -HGHGHG) histidine residues (Cong et al., 2012; by the conjugation of drugs or different organic molecules. As a repre­ Peciak et al., 2019). sentative example, in the Antibody Drug Conjugates (ADCs) the thera­ peutic molecule is covalently attached to a protein nanocarrier for its 5.4. Generation of cyclic ketone targeted delivery. Usually, protein nanomaterials are also linked to different organic molecules such as Polyethylene Glycol (PEG) to The use of an electrophile molecule such as 2-cyclohexanone has also improve their pharmacokinetic properties, stability or immunogenicity. allowed cation-free site-selective modification of surface-accessible In all cases, site-directed conjugation is very convenient, not only histidine residues on a protein. This Michael addition generates a cy­ because it would allow molecular binding in a desired and controlled clic ketone able to form an oxime by the addition of aminooxy-derivative position of the protein, but also since such strategies generate more probes. Using this methodology, different types of molecules including homogeneous products with low batch-to-batch variability and with nuclear magnetic resonance (NMR) tags, fluorescent dyes or biotin have higher stoichiometric control. In this sense, engineering of histidine-rich been successfully attached to a His-tagged protein (Joshi and Rai, 2019). motifs within the protein also plays an important role as site-specific conjugation targets. 5.5. Histidine acylation 5.1. Divalent transition metal ion coordination Finally, in a similar approach, specific acylation of an optimized amino-terminal histidine tag (GHHHn) has been also reported using D- As the most explored approach, many molecules chelated to divalent gluconic acid δ-lactone (GDL) and 4-methoxyphenil esters. This His-rich transition metal ions such as Ni2+ or Zn2+ have been attached to tag specific acylation methodology has allowed the introduction of small different nanomaterials (Table 1). This is done by the generation of reactive groups such as azides in different proteins including GFP, ternary complexes with the unpaired electrons in imidazole rings of maltose-binding protein (MBP) or the small ubiquitin-like modifier specific histidine residues as previously described. In this sense, NTA- protein (SUMO) for further reaction with DBCO-functionalized mole­ modified DNA molecules have been conjugated to proteins such as cules or direct conjugation of biotin (Martos-Maldonado et al., 2018). Green Fluorescent Protein (GFP) (Goodman et al., 2009) or alkaline phosphatase (Shimada et al., 2008) using Ni2+ ion coordination as a 6. Refined protein assembling and functional protein molecular glue. This strategy has been also used to attach different types aggregation of molecules such as monophospharyl lipid A adjuvants containing nanolipoprotein particles (MPLA:NiNLP) to His-tagged foot-and-mouth Artificial but largely bioinspired nanostructured materials have been disease virus capsids (Rai et al., 2016), or NTA-modified quantum dots developed based on protein building blocks, with high precision and to progeny virus capsids (Zheng et al., 2016). Further, NTA-modified structural complexity, integrating order and dynamics into the system gold-polymer nanoparticles have been also conjugated to His-tagged (Hamley, 2019; Korpi et al., 2020). Among the different protein-based antifreeze peptides to generate new active nanomaterials (Wilkins assembling approaches, simple fusion protein engineering permits the et al., 2019). incorporation of peptidic cross-interactors and the facile synthesis of de novo multimeric nanomaterials. This is done by bottom-up fabrication 5.2. Templated addition of alkynyl group or lysine conjugation approaches that ensure a control over the range of biochemical (e.g. specificity, bioactivity or biodegradability) and physical (e.g. small size, Hexahistidine tags can be also used as templates for a first-step conformation or multivalency) properties (Mendes et al., 2013). His-rich temporal cation-coordinated molecular binding and subsequent cova­ tags in recombinant fusion proteins have been empirically tested to lent bonding with a neighboring histidine residue within the protein. In construct regular, stable and highly versatile nanocarriers with pseu­ this sense, templated alkylation of an imidazole residue in a H6 tag can dospherical architectures (Lopez-Laguna et al., 2019), highly suited for be achieved using Baylis-Hillman esters tethered to NTA in presence of precision intracellular drug delivery (Falgas et al., 2020; Pallares et al., divalent cations. Here, Ni2+-coordinated ternary complex is generated 2020). One of the most exploited chemical mechanisms that drives the between NTA and two adjacent histidine residues. This event brings the assembling process based on H6 tags is the ion-based gluing (Lopez- reactive double bond of the Baylis-Hillman ester into the vicinity of Laguna et al., 2020). In this regard, divalent cations (e.g. Ni2+, Mg2+, another free imidazole ring for its covalent binding and subsequent Cu2+, Co2+, Zn2+, and Ca2+) are able to simultaneously coordinate breaking of the previous NTA bond. This methodology allows the more-than-one histidine residues from overhanging histidine tails, 7 H. López-Laguna et al. Biotechnology Advances 54 (2022) 107817 promoting building block recruitment and adequate protein organiza­ et al., 2013), which still allows an efficient purification through tion at the nanoscale (Fig. 3D). This process, usually renders materials immobilized metal affinity chromatography (Knecht et al., 2009). Be­ ranging between 10 and 350 nm, which are efficiently disassembled by sides this, the influence of the position of the tag at the carboxy or amino EDTA (Lopez-Laguna et al., 2020). The gluing process takes place when terminal end of the protein has been studied (Hofstrom et al., 2013; the respective ion interacts with the nucleophilic electron pair from δ1 Hofstrom et al., 2011; Tolmachev et al., 2010; Vorobyeva et al., 2019) nitrogen of the histidine imidazole ring and accommodates at least 2 by using radioactive labeling, to determine their organ biodistribution in near-in-space building blocks into the final oligomeric structure. These mouse models. The resulting data indicated that variations in the body interactions could also take place when carboxyl groups from asparagine localization of the materials do not rely exclusively on the histidine tail, and glutamine amino acid residues are close by (Erthal et al., 2016; but also on the presence of charges and hydrophobic patches on the Lopez-Laguna et al., 2019). When envisaged for biomedical applica­ scaffold protein and the way they are distributed (local vs. overall) on tions, engineering ionic doses should be appropriately adjusted to avoid the protein surface (Hofstrom et al., 2011; Lindbo et al., 2016; Tol­ possible systemic toxicity as stated in the recommended dietary allow­ machev et al., 2010). Even though the different studies are not compa­ ance (RDA) (Lopez-Laguna et al., 2020). A huge variety of different H6- rable due to the use of different chimeric scaffold proteins such as tagged materials have been lately constructed (Table 1) with different affibodies (Altai et al., 2016; Hofstrom et al., 2011; Mitran et al., 2015; morphologies and pointing to different applications (Fig. 3E). Tolmachev et al., 2010), nanobodies (Singh et al., 2014), scFvs (Casey In this sense metal-controlled protein dimers have been generated et al., 1995), toxins (Altai et al., 2016), uniCAR target modules (Jurec­ upon zinc binding to designed histidine residues (Maniaci et al., 2019), zek et al., 2019), ADAPT scaffolds (Lindbo et al., 2016), DARPIN scaffold which in some particular cases such as CheA/W chemoreceptors can (Vorobyeva et al., 2019) and also due to different linker chemistry for further assemble into ternary complexes to generate functional arrays radioactive tagging (Lindbo et al., 2016), it can be globally inferred that (Haglin et al., 2017). The bioengineering of strategically placed metal- hydrophobic domains and positively charged areas favors liver uptake coordinating histidine residues has also allowed the generation of syn­ (Altai et al., 2016; Hofstrom et al., 2013; Mitran et al., 2015; Singh et al., thetic coiled coil structures (Tunn et al., 2018). Going still further, the 2014; Tolmachev et al., 2010). In this sense, His-based tails like (HE)3, combination of specifically oriented metal coordinating histidines with hydrophilic and negatively charged, could help to counterbalance the additional intrinsic non-covalent interactions has allowed the genera­ hydrophobicity and positive charges of the area (Hofstrom et al., 2013; tion of more sophisticated nanostructures. These include linear nano­ Hofstrom et al., 2011; Mitran et al., 2015; Tolmachev et al., 2010), while wires of different lengths (Zhang et al., 2012), circular nanorings whose (HT)5, uncharged, gets mostly cleared by kidneys (López-Laguna et al., diameters can be regulated by tuning the strength of the non-covalent 2019). Also, poly-histidine tails at the amino terminal end show, in interactions (Bai et al., 2013), or even multi-responsive triblock copo­ general, a low level of nonspecific organ uptake, especially in kidney lypeptides that self-assemble as 20 nm diameter nanocapsules (van (Hofstrom et al., 2013; Hofstrom et al., 2011; Vorobyeva et al., 2019). In Eldijk et al., 2016). Other sophisticated structures, generated through contrast, other studies show that the histidine tail does not affect metal-histidine interaction, also include helical nanotubes, whose significantly the biodistribution of the protein (Casey et al., 1995; widths can be controlled by solution conditions (Brodin et al., 2015), Jureczek et al., 2019; Lindbo et al., 2016), indicating again that changes stimuli-responsive interfacial films (Dexter et al., 2006) or toroid of the protein fate in the body depend more on the overall distribution of nanoparticles (e.g. toxin-based (Diaz et al., 2018; Serna et al., 2020a), charged and hydrophobic amino acids of the His-tagged protein than on venom-based (Serna et al., 2020c) GFP-based (Cespedes et al., 2018; the mere presence and nature of the His-rich tag (López-Laguna et al., Pallares et al., 2020; Unzueta et al., 2020) or human nidogen-based 2020). Of course, all these concepts and considerations have important (Alamo et al., 2021), either being intrinsically toxic or delivering implications in the therapeutic applications of His-tagged proteins. coupled antitumoral drugs (e.g. floxuridine or monomethyl auristatin E). Finally, in a step further regarding structural complexity, functional 7.2. Intracellular trafficking secretory-like granules at the micro scale have been generated also through controlled protein aggregation via H6 tag–divalent cation co­ His-rich tags have been exploited as endosomal escape enhancers to ordination. These artificial aggregates permit the slow release of a deliver proteins or nucleic acids to the cytosol due to their ability to protein drug in a prolonged manner, then acting as a sustained drug trigger osmotic swelling and endosomal disruption (Ferrer-Miralles release systems aimed at reducing the need of repeated doses (Chen et al., 2011). Since the pKa of their lateral chain imidazole group is et al., 2019; Jiang et al., 2011; López-Laguna et al., 2021; Sanchez et al., around 6.0, histidine residues undergo protonation state change from 2020; Serna et al., 2020b). neutral to positive when endosomal lumen becomes acidic. In conse­ quence, not only H+ protons enter the lysosomes, but Cl− anions also do 7. Systemic administration for therapeutic applications it likewise in order to equilibrate charges, which generates an osmotic pressure that ends up with membrane disruption, namely the proton When proteins with histidine tails are meant to be used for sponge effect (Fig. 3F). Following this mechanism, His-tagged proteins biomedical purposes, it is essential to know whether histidine tails could can promote higher cytosolic delivery of their cargo molecule (e.g. change the behavior of therapeutic proteins when they are applied in nucleic acids and other drugs (Ferrer-Miralles et al., 2011)) into the vivo. It is especially critical to study whether histidines play a role in cytosol. Also, based on their selective protonation at acidic pH, histi­ protein biodistribution as well as subcellular localization. To this pur­ dines has resulted attractive in the design of novel endosomolytic pep­ pose, different clarifying studies have been carried out, the results of tides (Boeckle et al., 2006; Paray et al., 2021). In this regard, the number which are described below. of histidine residues, their distribution in the primary sequence and the endosomal vesicle size and membrane leakiness, has been described to 7.1. Biodistribution affect this process (Ahmad et al., 2019; Kichler et al., 2007; Lo and Wang, 2008; López-Laguna et al., 2020; Vermeulen et al., 2018). Because of their universal use as purification tags, the potential (Munsell et al., 2016; Smith et al., 2019). Therefore, His-rich tag- impact of His-rich tails in the biodistribution of systemically adminis­ dependent proton sponge effect can be considered a lysosome-specific tered proteins and their implication in drug delivery have been carefully membrane disruption system, although its real impact in promoting explored in different experimental settings. In these studies, the role of endosomal burst remains still controversial the amino acid composition of the histidine-rich tail has been evaluated, using intercalated hydrophobic or hydrophilic amino acids with different charges and upon an H-X-H-X-H-X arrangement (Hofstrom 8 H. López-Laguna et al. Biotechnology Advances 54 (2022) 107817 8. Bottlenecks associated to polyhistidine tags occasionally observed upon His-rich tag fusion (Woestenenk et al., 2004). Because of the chemical versatility of His-rich tags, a plethora of The variability of the already presented conformational scenario different functional materials and surfaces have been constructed opens up to the idea of using small peptide linkers to reduce the following bottom-up rational approaches. In this sense, although no conformational impact of fusion his-rich tags and to ensure an adequate intrinsic signs of cytotoxicity has been reported in the literature for conformational structure thus refining the design of bioactive fusion polyhistidine tags, its potential influence over the protein structure, proteins. In this sense, three major categories of linkers can be found stability or function should be precisely considered when designing according to their physicochemical or functional properties (flexible, devices for biotechnological or biomedical applications (Zhao and rigid or in vivo cleavable), which can be selected depending on the Huang, 2016). Interestingly, while B-factor values (reflecting thermal particular scientific needs. In fact, these linkers could not only motion) were usually slightly higher in His-tagged proteins, no signifi­ contribute to minimize the conformational impact of introduced tags cant structural effects have been generically observed neither in both over the protein but also improve the biological properties of the tagged resolution or R-factor (reliability factor) when considering a broad protein by increasing their production yields, by achieving desirable spectrum of crystallographic 3D protein structures with or without fused pharmacokinetic profiles or by improving their biological activity. In His tags (Carson et al., 2007). Similar results were obtained in recom­ this regard, flexible linkers appear as the most appropriate ones to binant GFP-based fusion proteins, in which H6 was placed in either the reduce the conformational impact of the histidine tag, as they confer amino or carboxy termini or in a permissive solvent-exposed loop of the sufficient mobility and flexibility to such tag. Flexible linkers are GFP barrel. In all cases, protein fluorescence remained intact and puri­ generally composed by small and either polar (e.g. Serine; Ser or Thre­ fication took place without any technical issues (Voltà-Durán et al., onine; Thr) or non-polar (Glycine; Gly) amino acid residues. Their small 2019). size allows the protein sequence being sufficiently mobile, and their polarity contribute to protein stability in aqueous solution not only by 8.1. Conformational impact forming hydrogen bonds with water molecules, but reducing the unfa­ vored interactions between other protein regions. The most commonly However, the influence of H6 tags on structural propensities and used flexible linkers include (Gly-Gly-Gly-Gly-Ser)n or (Gly-Ser)n se­ internal dynamics of small peptides might be more noticeable. While quences. In this sense, the n value could be adjusted for an optimized small peptides are highly mobile with no unique preferred spatial separation between functional domains, or to maintain necessary inter- conformation, the addition of certain peptides such as His-based tags, domain interactions. Although Gly and Ser are the most used amino triggers small chemical shifts and changes relaxation properties, being acids, Thr and Alanine (Ala) could be also introduced to maintain suf­ translated into long-range effect on overall structural propensities ficient structural flexibility and Lysine (Lys) and Glutamine (Glu) to (Bräuer et al., 2019). This possibility was demonstrated when studying improve sequence solubility. In all cases, the rational design should be the His-rich tag effects on zinc finger proteins, in which the addition of supported by experimental data, ensuring the optimization of the linker those peptides either at their amino or carboxy-terminal positions pro­ sequence, in order to fit with the particular needs linked to the recom­ moted changes in their native or complexed-with-zinc CD spectra, binant protein (Argos, 1990; Chen et al., 2013). unfolding profiles under urea degeneration tests, and protein function­ ality (Zhao and Huang, 2016). Alike influences were observed in di­ 8.2. Immunogenicity sulfide bond structures but not in the binding behavior of the rat corticotropin-releasing factor receptor 2a in a His-rich tag carrying On the other hand, immunogenicity assessment is paramount to version (Klose et al., 2004). In this regard, while a huge catalogue of ensure the safe and effective development of therapeutic proteins (Ishii- proteins should not be nearly affected by His-rich tag fusion, enzymes Watabe et al., 2018), since mitigation or full ablation of immunological- appear as a special and particular group sensitive to small structural based adverse events such as anaphylaxis, cytokine release syndrome, changes by either creating or depleting functional scenarios. In this and cross-reactive neutralization of endogenous proteins is mandatory context, the carboxy-terminal tagging of tropinone reductase enzyme (Lau and Dunn, 2018; Tourdot and Hickling, 2019). Indeed, immuno­ impaired its enzymatic activity while the amino-terminal tagged version genic responses correspond to the most common non-desired side effects remained fully active (Freydank et al., 2008). In agreement with that, in of proteins in clinics. In this scenario, protein-based drugs are currently silico modelling of the protein structure showed an interference of the transforming the pharmaceutical industry, displaying an unpreceded H6-tag with the enzyme active site by steric and electrostatic in­ potential specially in precision medicines (Sanchez-Garcia et al., 2016; teractions only when placed at the carboxy terminal (Freydank et al., Serna et al., 2018) and promoting the development of highly effective 2008). Similar scenarios were also described in L-lactate dehydrogenase, treatments (Lagasse et al., 2017). Then, immunogenicity of potential in the molybdoenzyme YedY, in secretory leukocyte protease inhibitors tags used in production might be a critical issue (Tourdot and Hickling, (SLPIs) and in hormone-sensitive lipases (HSL), in which the carboxy- 2019). From here, a dichotomy appears; while a large catalogue of terminal (Halliwell et al., 2001; Munadziroh et al., 2020; Sabaty et al., manufactured proteins with clinical potential are His-tagged, a public 2013) or the amino terminal (Wang et al., 2019) tagging resulted in concern shows up in which the use of H6 tag is discouraged by the Food reduced activities. The H6 tag fusion can also elicit a modulatory in­ and Drug Administration (FDA) and the European Medicines Agency fluence on the enzymatic activity of certain proteins and be used (EMA) due to its potential non-desired immune response, although no therefore as an advantage, as observed in the catalytic capacity of the official specifications are publicly indicated. In this context, alternative nuclease domain of the Colicin E7 metallonuclease (NColE7) from E. histidine-rich tags have been designed and tested with the aim of solving coli, an enzyme used by E. coli to kill competing bacteria under stress these regulatory concerns. For that, humanized versions, alternative to conditions (Abd Elhameed et al., 2020). In this regard, the interference the traditional H6, were constructed derived from BLAST-ed human of the oligohistidine peptide tag with the Zn2+ containing active center proteins namely H3A (HAAHAH), H5T (HTHTHTHTH) and H5E was used for the regulation and fine tuning of the enzymatic activity. (HEHEHEHEH). Interestingly, all humanized His-tag versions main­ From another point of view, scanning fluorimetry profiles displayed tained their protein production, purification and functional properties, both beneficial and deleterious effects on the thermal stability of the displaying a huge potential to be used in humanized biomedicines protein, depending on the presence or absence of a H6 in the amino- (López-Laguna et al., 2020). terminus (Booth et al., 2018) possibly due to an occasional architec­ From a technical point of view, part of the scientific community tonic rearrangement of the final protein structure. Mild and case- considers H6 sequences as safe tags, due to its small size (0.84 kDa), associated modulation of protein solubility have been also absence of electric charge (non-interactive material), and low cell 9 H. López-Laguna et al. Biotechnology Advances 54 (2022) 107817 toxicity and immunogenicity (Loughran et al., 2017). This is of course in observed at alternative increasing number-of-histidines His-tags, addition to the very convenient potential of His-rich segments as puri­ namely H-His-NH2, H-His6-NH2 and H-His12-NH2. This was indicative fication tags, at low costs and under mild imidazole-promoted elution of the irrespective influence of the number of close histidines into the and native or denaturing conditions (Lau and Dunn, 2018; Loughran final catalytic capacity of the tag. Finally, in a further step, the addition et al., 2017), making feasible the industrial production of protein drugs. of a His-tag into an enzyme structure can generate additional functions Notably, protein BLASTs also shows coincidences in more-than-six (dual-active enzymes) as reported in a His-tagged phenylacetone mon­ consecutive histidine segments found in human proteins, hinting that ooxygenase (His6-PAMO) able to hydrolyze p-nitroacetophenone to p- human immune system is not expected to react when administering H6- NP (via p-NPA), apart from its intrinsic natural oxidoreductase activity tagged formulations (López-Laguna et al., 2020). Besides this conceptual (from phenylacetone to benzyl acetate). Interestingly, this gain of consideration, there is no huge quantity and detailed experimental facts function was inhibited in presence of a molar excess of NiCl2, which was describing H6 immunogenicity, neither in vivo nor in vitro. In addition, able to specifically bind to the imidazole ring and block its catalytic His-rich tags have been included in multiple vaccine candidates against reactivity (Schoonen et al., 2017). malaria (Angov et al., 2003; Dutta et al., 2002; Otsyula et al., 2013), Because of that, the catalytic propensity of his-tags must also be proceeding as far as phase IIb in clinical trials without apparent safety considered when designing novel protein-based materials, to address concerns in vaccinated children (Jin et al., 2017). A similar scenario was undesired scenarios of gain of function. observed in tandem phase I studies evaluating reactogenicity, safety and immunogenicity of His-tagged antigens (Plasmodium falciparum surface 9. Concluding remarks protein; MSP142) administered intramuscularly with AS01 as adjuvant (Otsyula et al., 2013). On the other way around, the amino-terminal His- Although short histidine-rich peptides were initially developed as tagged Streptococcus pneumoniae surface protein SP08456 promoted tags for recombinant protein purification, their pleiotropic nature has undesired immunogenic responses when administered intramuscularly, facilitated their progressive and expansive adaptation as functional and in comparison to its non-tagged version for bacteria immunization in structural agents in multiple biotechnological and biomedical fields that Balb/c mice (Singh et al., 2020). In short, there are not experimental are sustained by protein-based materials. They range from catalysis to concluding remarks that support or preclude the use of His-tagged drugs drug development, but include dozens of different refined uses such as into clinics. designing advanced smart micro- and nano-scale materials, tools for precision medicines or for fine diagnosis approaches. Such innovative 8.3. Catalytic activity of imidazole rings applications open exciting possibilities to tailor proteins as powerful devices with unexpected regulatable properties, as purpose-tailored The addition of His-tags in recombinant proteins for purification or functional devices. other purposes may not be as innocuous as previously thought. Imid­ azole rings appear to be reactive species able to catalyze specific enzy­ Declaration of competing interest matic reactions, and so changing the resulting properties of the His- tagged protein material. The authors declare that there is no conflict of interest. In this sense, the catalytic function of the imidazole ring has been reported since 1956 in several scenarios, in which different types of Acknowledgments polymers, nanoparticles, peptide bundles and nanofibers have been involved (Bai et al., 2011; Chadha and Zhao, 2013; Giusti et al., 2014; The authors are indebted to Agencia Estatal de Investigación (AEI) Guler and Stupp, 2007). Indeed, this chemical group has been described and to Fondo Europeo de Desarrollo Regional (FEDER) BIO2016-76063- as able to catalyze the transformation of p-nitrophenyl acetate hydro­ R, AEI/FEDER, UE to A.V. and PID2019-105416RB-I00/AEI/10.13039/ lysis (p-NPA) (Denmark et al., 1990), as well as, having a capital role in 501100011033 to E.V., AGAUR 2017SGR-229 GRC to A.V., CIBER-BBN aldol-based and RNA cleavage reactions (Anslyn and Breslow, 1989; (projects NANOPROTHER and VENOM4CANCER to A.V., NANO­ Breslow, 1994; Kirby and Marriott, 1995; Markert et al., 2009; Scheffler REMOTE to E.V. and NANOSCAPE and NANOLINK to U.U.), and to and Mahrwald, 2012). Its chemical versatility (especially when con­ Instituto de Salud Carlos III (ISCIII) PI20/00400 co-funded by European forming the side chain of the histidine amino acid) lies in its amphoteric Regional Development Fund (A way to make Europe) to U.U., for their nature. Thus, the imidazole ring is able to lose a proton via its pros ni­ research in protein production and in functional protein materials. The trogen atom or to accept a proton via its tele nitrogen atom at pHs close authors are also indebted to CERCA programme from la generalitat de to 7 (Schneider, 1978). Because of that, histidine residues are suitable Catalunya, Spain and to the Networking Research Center on Bioengi­ for both nucleophilic and base catalysis reactions, in addition to the neering, Biomaterials and Nanomedicine (CIBER-BBN), Spain that is an traditional metal coordination functions. initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Therefore, due to this intrinsic chemical reactivity of the imidazole Ingenio 2010, Consolider Program, CIBER Actions and financed by the ring, His-tags might be not biologically inert when fused to a protein. Instituto de Salud Carlos III, with assistance from the European Regional Among the different experimentally described scenarios, the His-tag can Development Fund. Molecular graphics and analyses were performed present esterase activity as reported in a GFP-H6 protein model, being with UCSF ChimeraX, developed by the Resource for Biocomputing, able to convert p-NPA to p-nitrophenol and acetic acid in a linear con­ Visualization, and Informatics at the University of California, San centration dependent way, and in an aqueous PBS-based solution Francisco, with support from National Institutes of Health R01- (Schoonen et al., 2017). This esterase catalytic activity was also GM129325 and the Office of Cyber Infrastructure and Computational observed with other esterase substrates such as p-nitrophenyl butyrate Biology, National Institute of Allergy and Infectious Diseases. H.L.L. was (p-NPB), polyethylene glycol MW 800-modifed p-NPA (p-NPA-PEG) and supported by a predoctoral fellowship from AGAUR (2019FI_B00352), carboxyfuorescein diacetate (CFDA). In addition, an increase in His-tag- and E.V.D. was supported by a predoctoral fellowship from Ministerio de mediated esterase activity was observed upon assembling of an His- Ciencia e Innovación, Spain (FPU18/04615). U.U. was supported by a tagged cowpea chlorotic mottle virus (CCMV), showing that the bigger Miguel Servet contract (CP19/00028) from ISCIII co-funded by Euro­ the protein-based structure, the higher the activity. The activity increase pean Social Fund (ESF investing in your future). A.V. received an ICREA upon protein assembling could be associated with a cooperative effect ACADEMIA award. between close-in-space imidazole rings of the His-tag from each protein building block. 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