To introduce ketones into the coat proteins, phage were transaminated using a 100 mM solution of pyridoxal 5’-phosphate (PLP) at pH 6.5 for 13 h. The excess PLP was then removed by precipitating the phage, after which they were exposed to various alkoxyamine compounds in pH 6.5 buffer for up to 24 h. Aniline catalysis was used to accelerate oxime formation, as has been previously reported by Dawson and coworkers.26
The specific reaction times and alkoxyamine concentrations were selected based on the levels of modification sought. To estimate the overall extent of p8 modification, a sample of ketone-labeled fd phage was reacted with 2-(aminooxy)acetic acid. Analysis of the coat proteins was achieved using MALDI-TOF mass spectrometry, revealing that the vast majority of the p8 proteins formed the oxime product (, Supporting Information Figure S1
), indicating that each fd phage can be loaded with thousands of molecules. Only one addition per p8 was observed, indicating N-terminal specificity even in the presence of five p8 lysines. The overall protein recovery for the transamination and oxime formation steps ranged from 55–95%, with 80% being a typical value. Unfortunately, despite many attempts, protein digest experiments failed to give any cleaved species for the p8 protein, presumably due to its very low solubility and propensity for aggregation once removed from the assembled structure.
Figure 2 Analysis of filamentous phage modified with small molecules. a) Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrum showing p8 oxime formation following reaction with 2-(aminooxy)acetic acid (expected mass increase: 73 m/z, (more ...)
The only by-product was a small amount of a covalent adduct of the protein with the PLP, which presumably formed through an aldol addition of the N-terminal pyruvamide to the pyridoxal aldehyde group. This PLP adduct was not visible following reaction of phage with aminooxy-derivatized molecules via
MALDI-TOF mass spectrometry analysis, possibly due to its poor ionization or insufficient quantity. It was, however, identified by mass spectrometry after disassembling the phage using RP-HPLC to isolate the PLP adduct-p8 from wt- and ketone-p8 species (Supporting Information Figures S2 and S3
). The negative charge of the phosphate group resulted in the earlier elution via
RP-HPLC. This species has been observed in transamination reactions previously, and since it possesses a ketone group, it can still participate in oxime formation.16
This, in addition to its very low abundance, renders it insignificant for most applications.
The small p3-to-p8 ratio for fd phage prevented p3 detection by mass spectrometry and western blotting. Instead, we turned to the use of M13KE phage, which are fd analogs with smaller genomes. They require a smaller number of p8 proteins to tile the length of the phage, and therefore have a higher ratio of p3 to p8 proteins. The M13KE and wt-fd coat proteins are identical, except for a single D12N point mutation in p8.27,28
To detect the modifications with improved sensitivity, transaminated M13KE was exposed to biotin-ONH2
and analyzed via
western blotting with neutravidin-HRP (). All of the coat proteins with accessible N-termini, including p3, showed labeling. The α-p3 blot in shows that both lanes contain approximately the same concentration of phage, while the neutravidin-HRP (α-biotin) blot shows that only PLP-reacted phage are biotin labeled.
To verify the ability of the modified phage to bind their targets, samples of transaminated anti-EGFR, anti-HER2, and anti-BoNT fd phage were reacted with Alexa Fluor® 488 or 647 C5-aminooxyacetamide (AF488/647-ONH2
) dyes. For the cell microscopy experiments described below, approximately 6–8% of the p8 proteins (~300 copies/phage, as determined using UV/vis) were labeled with the fluorophores. We have increased the levels of fluorophore modification with AF488 to levels of up to 80% by using a ten-fold excess of aniline26
(100 mM for AF488), albeit with some levels of decreased solubility. The modified phage bound to their appropriate cell surface receptors with excellent specificity, as revealed using flow cytometry ( and Supporting Information Figures S4–S7
). The negative control anti-BoNT phage showed no binding. In terms of cell viability, these data also indicated that only 0.25% to 3.0% of the cells had died during the exposure to the phage-based imaging agents, which was in line with untreated cell samples.
Figure 3 Fluorophore modified fd phage cell binding results. a) Flow cytometry with AF488 labeled phage (applied at 0.8 nM) indicated selective recognition of EGFR and HER2 epitopes. The legend for all histograms is shown in SUM52PE inset. Gating data are shown (more ...)
The selective binding capabilities of the EGFR and HER2 targeted phage were also confirmed in microscopy experiments. A panel of breast cancer cells was treated with the phage, and visualized using live cell confocal microscopy. These images ( and Supporting Information Figures S8–S12
) demonstrated the retention of excellent specificities and binding capabilities of fd for their targeted receptors following chemical modification. Upon increased incubation times (>2h), phage targeting overexpressed markers were observed to be internalized by the respective cells. Preliminary results indicate that this occurs via
receptor-mediated endocytosis; however, further experiments to clarify this behavior are in progress.
The ability of these fd to image receptor overexpression in vitro
, even when different cell types are mixed, portends well for their use in vivo
. In anticipation of future in vivo
applications, we investigated the attachment of poly(etheylene glycol) (PEG) polymers to the phage capsids. PEG has been shown to reduce non-specific binding, decrease immunogenicity, and increase the solubility of attached molecules.29
Ketone-labeled fd were reacted with 2 kDa O
-(methoxypoly(ethylene glycol))-hydroxylamine (PEG2k-ONH2
and the percentage of p8 proteins that were modified was quantified using RP-HPLC (Supporting Information Figure S13
). By varying the reaction times, samples with differing levels of PEG2k-labeled p8s were prepared. Presumably higher concentrations of the PEG2k-ONH2
could achieve shorter modification times, but we avoided using them to prevent precipitation of the phage (as was purposely done in the protein purification steps). For phage previously labeled with fluorophores, there were no observed changes in the absorption or emission properties of the dyes upon addition of the chains. The added PEG chains also caused no morphological changes that could be observed by TEM (Supporting Information Figure S14
Zeta potential measurements were obtained in order to determine the ability of the PEG polymers to shield fd charge (Supporting Information Figure S15
). An increase in negative charge was noted following PLP modification, presumably due to the loss of the cationic N-terminal amino groups on the p8 monomers. As anticipated, the negative charge decreased with increasing levels of PEG modification. At 67% p8 labeling the zeta potential was −5.5±7.3 mV, nearly an order of magnitude less than that of ketone-labeled fd. The binding abilities of PEG-labeled fd were also evaluated by flow cytometry ( and Supporting Information Figures S16–S18
). The PEG-labeled anti-EGFR fd continued to bind MDA-MB-231 (EGFR positive) cells, while none bound the MCF-7 cl18 cells (EGFR negative).
Figure 4 Flow cytometry analysis of AF488 labeled anti-EGFR fd possessing various levels of PEG modifications. The target cells were MDA-MB-231 (EGFR+, left) and the control cells were MCF-7 cl18 (EGFR−, right). Phage concentrations were 0.8 nM. Gating (more ...)