Directed Evolution and Engineering of Gallium-Binding Phage Clones-A Preliminary Study

Abstract

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-target interaction based on peptide structure and amino acid position and composition via the newly established systematic cysteine scanning approach.

Keywords: cysteine; gallium; metal–peptide interaction; peptide structure; phage surface display; site-directed mutagenesis.

Figure 1

Biosorption of gallium (A) and arsenic (B) to original gallium-binding phage clones and wild-type phage control at pH 8.5 in phosphate buffered solution and pH 3.2 in acetate buffered solution. Results are expressed as the amount of sorbed gallium (mg) to 10¹⁰ phage particles (pp). Error bars represent the standard error.

Figure 2

Relative binding affinity of original gallium-binding phage clones for gallium compared to arsenic at pH 8.5 in phosphate buffered solution and at pH 3.2 in acetate buffered solution. Results are expressed as the fold increase above the biosorption of wild-type bacteriophage particles. The values were normalized to the wild-type phage affinity for gallium compared to arsenic (Wt = 1). Error bars represent the standard error.

Figure 3

(A) Biosorption of gallium and (B) arsenic to mutant gallium-binding phage clones, original clone C3.129, and wild-type phage control at pH 8.5 in phosphate buffered solution and pH 3.2 in acetate buffered solution. Results are expressed as the amount of sorbed gallium (mg) to 10¹⁰ phage particles (pp).

Figure 4

Relative binding affinity of mutant gallium-binding phage clones for gallium compared to arsenic at pH 8.5 in phosphate buffered solution and at pH 3.2 in acetate buffered solution. Results are expressed as the fold increase above the biosorption of wild-type bacteriophage particles. The values were normalized to the wild-type phage affinity for gallium compared to arsenic (Wt = 1). Error bars represent the standard error.