Bacteriophage Engineering for Improved Copper Ion Binding

Abstract

In this study, fd viruses are genetically modified to display seven cropped versions (H, HG, HGF, HGFA, HGFAN, HGFANV and HGFANVA) of the previously identified Cu(II) specific peptide (HGFANVA). Atomic force microscopy (AFM) imaging reveals the typical filamentous structures of recombinant phages with thicknesses of ≈2–5 nm in dry state. Scanning electron microscopy (SEM) imaging shows that HGFANVA viruses form larger elongated assemblies than H viruses that are deposited with a mineral layer after Cu(II) treatment. C and N peaks are detected for virus samples through Energy dispersive X‐ray spectroscopy (EDX) analyses confirming the presence of phage organic material. Cu peak is only detected for engineered viruses after Cu(II) exposure. Enzyme‐linked immunosorbent assay (ELISA) analyses show the selective Cu(II) binding of engineered phages. Agarose gel electrophoresis (AGE) and zeta potential analyses reveal negative surface charges of engineered viral constructs. Positively charged Cytopore beads are coated with bacteriophages and used for Cu(II) ion sorption studies. ICP‐MS analyses clearly show the improved Cu(II) binding of engineered viruses with respect to wild‐type fd phages. Such bottom‐up constructed, genetically engineered virus‐based biomaterials may be applied in bioremediation studies targeting metal species from environmental samples.

Reprogramming Filamentous fd Viruses to Capture Copper Ions

“…A certain degree of Cu(II) binding with respect to other metal ions could be observed for all virus constructs (Figure 9). However, C‐terminal truncated variants showed considerably lower Cu(II) binding than the full HGFANVA construct and the already reported N‐terminal cropped variants [24] . Since the N‐terminal cropped variants (H, HG, HGF, HGFA, HGFAN and HGFFANV) were all carrying one H unit, an enhanced Cu(II) binding was reasonable [24] .…”

“…Similar disordered and intertwined virus layer formation was reported by Yoo et al ., (2006) for M13 viruses, adsorbed from a highly concentrated virus solution onto a polymeric surface [28] . Height profiles of protruding virus filaments for each construct are included in S2 showing a dry state thickness of around 2 nm as previously reported [24] …”

“…In our previous study, seven N‐terminal cropped peptide variants (H, HG, HGF, HGFA, HGFAN, HGFANV and HGFANVA) of the Cu(II) specific peptide HGFANVA (Cu5 peptide [22] ) were expressed on the body part of fd phages through site directed mutagenesis [24] . In that study, we have demonstrated that the display of a single H unit enhanced Cu(II) binding of the engineered viruses with respect to wild type fd phages.…”

“…C‐terminal cropped variants (A, VA, NVA, ANVA, FANVA and GFANVA) of our previously identified Cu(II) specific peptide HGFANVA (Cu5 peptide) [22] were displayed on >2500 copies of P8 major coat proteins. P8 display of these peptides was performed by inserting each peptide coding DNA sequence into the 5‘flexible region (AEGDD) of fd‐tet vector backbone (9183 nucleotides, GenBank: AF217317.1) as previously described [24] …”

“…Both peptides were applied as biorecognition units to validate a colorimetric metal ion detection technique using AuNPs [22–23] . In our recent study, we generated genetically modified fd virus constructs displaying N‐terminal cropped versions (H, HG, HGF, HGFA, HGFAN, HGFANV and HGFANVA) of Cu5 peptide where we reported on the significant role of H unit on Cu(II) binding affinities [24] . In this study, we generated recombinant viruses displaying C‐terminal truncated variants (A, VA, NVA, ANVA, FANVA, GFANVA) of Cu5 peptide excluding the H unit.…”