Orthogonal Labeling of M13 Minor Capsid Proteins with DNA to Self-Assemble End-to-End Multiphage Structures

M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multiphage devices requires precise control of interactions between the M13 capsid proteins. Toward this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi-material devices

Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificity

Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificit

Dissociation of the C-terminal chaperone in the course of intoxication.

<p>HeLa cells were incubated with ^TAMRAAeL.CP^AF647 for 30 minutes at 4°C, washed, and the temperature shifted to 37°C. Images were acquired by confocal microscopy.</p

Strategies for site-specific labeling of proaerolysin.

<p><b>A</b> Structure of the proaerolysin monomer (PDB: 1PRE). Proaerolysin consists of several different domains, two of which are responsible for receptor binding (domains 1 and 2), one containsing the trans-membrane domain, and the C-terminal peptide (CP), which functions as a chaperone and dissociates from the rest of the complex upon heptamer association and pore formation. <b>B</b> Sortase reaction mechanism. C-terminal sortagging: sortase cleaves after threonine in the context of its recognition motif resulting in the formation of a new covalent bond with the N-terminus of an added oligoglycine or oligoalanine nucleophile coupled to a label of choice. N-terminal sortagging: the N-terminal glycine of proaerolysin is recognized as a nucleophile by sortase and conjugated to an LPXTG/A probe bearing a label. <b>C</b> Structures of probes used in this study. Not depicted is AAA.Alexa Fluor 647, which is similar to GGG.Alexa Fluor 647, but with alanine replacing glycine. PelB: periplasm targeting sequence, cleaved off by the producer bacteria upon export of proaerolysin to the periplasm. H6: hexahistidine handle for affinity purification. Protease cleavage sites are recognized by target cell surface proteases such as furin. CP: C-terminal peptide, serves as a chaperone for proaerolysin. Upon its loss, proaerolysin is converted to mature aerolysin (AeL). <b>D</b> Scheme for wild type (WT) and sortaggable versions of proaerolysin with their designations. The LPXTG/A pentapeptides are sortase recognition motifs.</p

Double-labeling of proaerolysin.

<p>Double-labeling was achieved with a two-step approach. <b>A</b> Schematic representation of the dual labeling strategy of proaerolysin. <b>B</b> We used SrtA_Strep to install an oligoalanine coupled to the fluorophore AF647 at the C-terminus of proaerolysin, followed by a gel filtration purification step. <b>C</b> Elution profiles were analyzed by SDS-PAGE, fluorescence scan and coomassie stain. <b>D</b> The reaction product was subjected to the second round of sortagging with SrtA_Staph7M and LPETG-coupled TAMRA fluorophore for N-terminal labeling. SrtA_Staph does not recognize or cleave LPXTA, hence the C-terminal label remains intact. A single peak is observed on the elution profile as immobilized sortase was used for the reaction and removed prior to gel filtration. <b>E</b> Elution profiles were analyzed by SDS-PAGE followed by fluorescence scan.</p

Impact of aerolysin modification on toxic activity.

<p>Aerolysin variants were titrated on KBM7 cells. 0.5×10⁵ cells per sample were incubated with toxin for 1 hour at 37°C in a total volume of 100 µL, stained with propidium iodide (PI), and the PI negative percentage determined by flow cytometry. The concentration range for the aerolysin variants ranged from 60 ng/mL to 4 pg/µL. Every condition was tested in triplicate. The percentage of PI negative controls was set to 100%, and the 50% lethal dose (LC50) calculated in R. 0.001 was added to all concentration values to avoid taking a log2 of 0.</p

Identification of new aerolysin receptors.

<p>^BiotinAeL.CP was used to identify new GPI-anchored proteins that bind Aerolysin<b>. A</b> Biotin.LPETG was attached to the N-terminus of proaerolysin via sortagging. The purified reaction product was analyzed by immunoblot. <b>B</b> HeLa cells were incubated with ^BiotinAeL.CP for 3 hours at 4°C and subsequently lysed with 0.5% NP-40. After pull-down with neutravidin beads, proteins were eluted, analyzed by SDS-PAGE, and subjected to mass spectrometry. Five GPI-anchored proteins were identified. UniProt accession codes are indicated. Peptides identified by mass spectrometry, lipidated amino acids, signal peptides, as well as peptides cleaved off from the pro-proteins are highlighted. <b>C</b> Binding of ^BiotinAeL.CP to mesothelin and to CD59 was verified by immunoblot.</p

Installation of a single label on proaerolysin.

<p>The fluorophore carboxytetramethylrhodamine (TAMRA) was installed at the N-terminus of aerolysin (^NAeL.CP), at the C-terminus of aerolysin upstream of the CP (AeL^C) and at the C-terminus of the C-terminal peptide (AeL.CP^C) with sortase. <b>A, C, E</b> Schematic representation of the sortagging reactions using of ^NAeL.CP, AeL.CP^C, AeL^C respectively. <b>B, D</b> Sortagging of ^NAeL.CP and AeL.CP^C, respectively, with respective control conditions, resolved by SDS PAGE and imaged with a fluorescence scanner. Product is visible by fluorescent signal. SrtA_Strep and SrtA_Staph recognize and cleave LPXTA and LPXTG motives, respectively. <b>F</b> Purification of labeled AeL^TAMRA, gel filtration. The first peak in the A280 elution profile corresponds to aerolysin, the second to sortase, and the third to free nucleophile. <b>G</b> Analysis of the first peak of the gel filtration elution profile with SDS PAGE followed by fluorescence image scan and Coomassie stain. A fraction of Ael^C is not converted to fluorescent product.</p

M13 Bacteriophage Display Framework That Allows Sortase-Mediated Modification of Surface-Accessible Phage Proteins

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool