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This study is concerned with a chromatography-based approach (Immobilized Metal Ion Affinity Chromatography) for the recovery of gallium binding peptide sequences from a recombinant phage display library. The here described methods apply the fundamental knowledge and methods of separation science and meet thereby the key requirement of the phage display technique of precise separation of target-binding bacteriophage clones from non-interacting bacteriophage during the biopanning. During the chromatopanning called process, a total of 101 bacteriophage clones were identified of which in subsequent binding experiments, phage clones expressing the peptide sequences TMHHAAIAHPPH, SQALSTSRQDLR and HTQHIQSDDHLA were characterized to bind >10 fold better to a target that presents immobilized gallium ions than control phage, displaying no peptide sequence. The performance of biopanning experiments in chromatographic systems is particularly suitable for demanding targets such as trivalent metal ions. We found, that the selection process benefits immensely from the stable immobilization of the target metal ions during the entire biopanning process as well as the complete recovery of well interacting bacteriophage clones. Among others, this was possible due to an enhanced monitoring of process conditions and fractionation of eluates.

Gallium (as Ga3+) is a Group IIIa metal and its recovery from wastewaters has become increasingly important for its reuse. The use of peptides for recycling offers a low-cost and environmentally-friendly option but the structural characteristics of peptides likely to bind Ga3+ are largely unknown. Multiple computational methods, coupled with experimental verification via NMR and Isothermal Calorimetry (ITC), were used to establish that Ga3+ binds with high affinity to peptide sequences and to elucidate the structural characteristics that contributed. It was demonstrated that peptide pre-organisation is key to Ga3+ binding and that a favourable binding position is necessarily governed by the size and shape of the electrostatic environment as much as individual electrostatic interactions with peptide residues themselves. Given favourable conditions, Ga3+ retrieved plausible binding positions involving both charged and uncharged residues that greatly increases the range of bonding possibilities with other peptide sequences and offers insights for binding other metals. The addition of pH buffer substantially improved the affinity of Ga3+ and a structural role for a buffer component was demonstrated.

Here we provide a proof of principle for an application-oriented concept for the peptide-based recovery of gallium in industrial wastewater, which was supported by biosorption studies with a real wastewater sample. We investigated the interaction of the gallium-binding peptides TMHHAAIAHPPH, NYLPHQSSSPSR, SQALSTSRQDLR, HTQHIQSDDHLA, and NDLQRHRLTAGP with gallium and arsenic through different experimental and computational approaches. Data obtained from isothermal titration microcalorimetry indicated a competitive influence by the presence of acetate ions with an exothermic contribution to the otherwise endothermic peptide gallium interactions. For peptide HTQHIQSDDHLA, a stabilizing influence of acetate ions on the metal peptide interaction was found. Peptide NYLPHQSSSPSR showed the highest affinity for gallium in ITC studies. Computational modeling of peptide NYLPHQSSSPSR was used to determine interaction parameters and to explain a possible binding mechanism. Furthermore, the peptides were immobilized on polystyrene beads. Thus, we created a novel and exceptionally robust peptide-based material for the biosorption of gallium from an aqueous solution. Data obtained from isothermal titration microcalorimetry indicated a competitive influence by the presence of acetate ions with an exothermic contribution to the otherwise endothermic peptide gallium interactions. For peptide HTQHIQSDDHLA, a stabilizing influence of acetate ions on the metal peptide interaction was found. Peptide NYLPHQSSSPSR showed the highest affinity for gallium in ITC studies. Computational modeling of peptide NYLPHQSSSPSR was used to determine interaction parameters and to explain a possible binding mechanism. Furthermore, the peptides were immobilized on polystyrene beads. Thus, we created a novel and exceptionally robust peptide-based material for the biosorption of gallium from an aqueous solution.

The development of effective and ecofriendly processes for the recovery of critical elements poses a challenge for scientists all over the world. A novel approach is the generation of highly specific peptides that bind with high affinity to individual elements of interest. The peptides are selected by phage surface display (PSD) technology. In this study, PSD technology has been applied in two different approaches. The focus of the first approach was the identification of peptides that bind specifically to special particles of interest that are part of electronic scrap aiming towards the development of new recycling processes. In the second approach, metal ion binding peptides were isolated via PSD to use them for the targeted removal and enrichment of these elements from complex leaching solutions or from industrial waters. To address the economic production of peptides, the development of a new expression system is also part of this study.

The phage surface display technology is a useful tool to screen and to extend the spectrum of metal-binding protein structures provided by nature. The directed evolution approach allows identifying specific peptide ligands for metals that are less abundant in the biosphere. Such peptides are attractive molecules in resource technology. For example, gallium-binding peptides could be applied to recover gallium from low concentrated industrial wastewater. In this study, we investigated the affinity and selectivity of five bacteriophage clones displaying different gallium-binding peptides towards gallium and arsenic in independent biosorption experiments. The displayed peptides were highly selective towards Ga3+ whereby long linear peptides showed a lower affinity and specificity than those with a more rigid structure. Cysteine scanning was performed to determine the relationship between secondary peptide structure and gallium sorption. By site-directed mutagenesis, the amino acids of a preselected peptide sequence are systematically replaced by cysteines. The resulting disulphide bridge considerably reduces the flexibility of linear peptides. Subsequent biosorption experiments carried out with the mutants obtained from cysteine scanning demonstrated, depending on the position of the cysteines in the peptide, either a considerable increase in the affinity of gallium compared to arsenic or an increase in the affinity for arsenic compared to gallium. This study shows the impressive effect on peptide&ndash, target interaction based on peptide structure and amino acid position and composition via the newly established systematic cysteine scanning approach.

Phage surface display (PSD) represents a powerful technique for isolating metal ion binding peptides. From a commercial pIII phage library, two specific peptide motifs—namely, CNAKHHPRC for nickel and CTQMLGQLC for cobalt—were identified using a specialized experimental set-up with sol-gel-coated glass fiber fabrics possessing ion exchange capacity. Employing single-clone binding experiments with immobilized Me2+ ions on nitrilotriacetic acid (NTA) agarose beads, adsorption isotherms were determined for both the phage clones and the reference clone without a peptide insert which clearly indicate specificity of the identified phage clones for their respective target ion. The mechanisms involved in phage binding to agarose beads could be satisfactorily described using the Freundlich model. In all experiments, two fractions of phages were reproducibly identified: those chemically eluted with glycine, and a second, smaller fraction that could be eluted only by direct incubation with the host organism E. coli ER 2738. The results demonstrate that metal ion-specific phage clones can be identified and characterized in terms of their binding strength with this experimental setup. In perspective, this approach can facilitate the identification of well suited phage clones for the further development of new element specific biosorptive materials for the recovery of heavy metals from dilute aqueous solutions, e.g. industrial waste waters.

Despite many innovations, meeting both economic and ecological requirements remains challenging for conventional resource recovery technology. The development of highly selective peptides puts a new competitor on the market. We present an approach to identify peptides for resource recovery using Phage Surface Display. Here, we describe the development of peptides for binding of rare earth element terbium-containing solids and for removal and enrichment of the heavy metal ions of cobalt and nickel out of waste waters and leaching solutions. We identified phage displaying specific peptides with ∼100× enhanced affinity towards terbium-containing solids or ∼20× enhanced affinity towards nickel (∼3× cobalt).

Phage display for discovery of metal binding peptides is an innovative way to engineer metalsorptive biological structures with a broad spectrum of applications in geobiotechnology, for example in reusable filter modules in biosorption processes. Using state-of-the-art cloning techniques we developed an easy-to-use cloning and expression system, allowing the fast production of identified peptides.

Phage surface display technology is a useful tool for the identification of biosorptive peptides. In this work it is used for the identification of cobalt, nickel and gallium binding peptides. We present methods for the enrichment of metal ion binding bacteriophage clones from two commercial phage display libraries. One of them presents cyclic heptamer peptides, in which two cysteins flanke the peptide loop (C7C), and a linear dodecapeptide library (D-12).Metal ion selective peptides are suitable to separate as well as concentrate cobalt and nickel from copper black shale leaching products (EcoMetals project) and gallium from industrial waste waters (EcoGaIn project). In contrast to common capture methods of specific binding phage for solid materials the ionic species have to be immobilized prior to the bio-panning procedure. This was realized by chemical complexation of the metal ions using commercial complexing agents on porous matrices. Moreover, an option to harvest non elutable strong binding phage is proposed.