Targeting mammalian organelles with internalizing phage (iPhage) libraries

Abstract

Techniques that are largely used for protein interaction studies and the discovery of intracellular receptors, such as affinity-capture complex purification and the yeast two-hybrid system, may produce inaccurate data sets owing to protein insolubility, transient or weak protein interactions or irrelevant intracellular context. A versatile tool for overcoming these limitations, as well as for potentially creating vaccines and engineering peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage-display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries using a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and for fingerprinting functional protein domains in living cells. Here we explain the design, cloning, construction and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ∼8 weeks.

Figure 1

Flowchart of the iPhage combinatorial library technology. Steps involved in iPhage construction, library cloning, and ligand/receptor validation are shown in bold. Times required to complete these steps are depicted in the time line (days). Pause points are indicated in red.

Figure 2

Generation of iPhage vector and library cloning strategy. The parental f88-4 phage vector contains two capsid genes encoding a wild-type protein VIII (pVIII, depicted in grey) and a recombinant protein VIII (rpVIII, depicted in green). The recombinant gene VIII contains a foreign DNA insert with a HindIII and a PstI cloning site, which allows the cloning of annealed oligonucleotides encoding the pen peptide in frame with the recombinant gene VIII. For generation of the iPhage vector, f88-4 displaying the pen peptide motif (RQIKIWFQNRRMKWKK) and fUSE5 phage plasmids are digested with BamHI and XbaI restriction enzymes, purified, and fused by standard ligation protocol. Next, an annealed random oligonucleotide library (e.g. X₄YX₄) is digested with the BglI restriction enzyme and cloned within the SfiI restriction site of the pIII coat protein gene (pIII, depicted in light blue) on the iPhage vector. MC1061 E. coli electro competent cells are transformed with the library cloned iPhage vector to produce a random peptide iPhage library. TetR – tetracycline resistance gene.

Figure 3

Overview of phage vector (f88-4, fUSE5, and iPhage) purification by CsCl. (a) Overnight bacterial culture is harvested and purified via a maxi-prep plasmid purification kit according to the manufacturer’s instructions ; a ‘clean phage plasmid’ prep is produced. For maximum phage plasmid purity (ultrapure), a CsCl/EtBr gradient is performed. After ultracentrifugation, the lower plasmid band (dsDNA band) is recovered and is precipitated by addition of 2-propanol into the DNA mix. 2-propanol will also remove the EtBr from the phage plasmid. Finally, addition of ethanol precipitates the DNA and removes any salt contaminants from the plasmid prep. (b) 0.8% E-gel analysis of fUSE5 purified with the Qiagen Maxi kit followed by CsCl/EtBr gradient purification (CsCl) or by Qiagen Maxi prep fUSE5 (Maxi) only. fUSE5 phage plasmid: 9206 Kb.

Figure 4

Systematic approach for affinity chromatography and receptor identification based on iPhage technology. Affinity columns are crafted by coupling of the selected synthetic peptide to agarose beads according to the manufacturer’s guidelines. Cell lysate is then loaded in the column and the receptor is eluted with the corresponding competitive peptide at a concentration of 5 mM. Eluted fractions are monitored by absorbance (optical density at 280 nm), desalted, and concentrated. Fraction screening is performed by the coating of equal amounts of protein on a 96-well plate and incubation with targeted iPhage or parental insertless iPhage (10⁹ TU each). Each phage population is recovered by host bacterial infection. Positive fractions (high number of bacterial colonies) are further analyzed by gel electrophoresis. Finally, unique bands are identified and selected for MS/MS analysis.

Figure 5

Overview of differential centrifugation, and subcellular fractionation quality analysis. (a) After an overnight incubation with the iPhage library, cells are recovered and transferred into a 15 ml conical tube. Cell pellet is washed and mixed with ice-cold hypotonic buffer. Cells are mechanically lysed with a Dounce homogenizer. Mitochondrial stabilization (MS) buffer is added to the cell lysate and after centrifuging the samples the supernate is recovered and transferred into clean microfuge tubes. The first pellet sample contains the nuclei, intact cells, and cell debri. Further fractionation may be performed to purify the nuclear fraction. Repeated centrifugation at progressively high speeds will separate cell components on the basis of size and density. (b) Equal protein concentrations were resolved in a 4-20% Tris-glycine gel and transferred onto a nitrocellulose membrane. Plasma membrane, cytosol, mitochondria/endoplasmic reticulum (ER), and nuclear fractions were tested with Cadherin (membrane marker), ERAB (mitochondrial marker), HMGB1 (nuclear marker), and actin (cytosol marker) antibodies.

Figure 6

iPhage organelle-targeting properties. (a) KS1767 cells were incubated with insertless Phage, insertless iPhage, and iPhage-NLS for 24 h at 37°C. Phage particles were recovered from mitochondria/ER and nuclear fractions, and phage homing was determined by bacterial infection and colony count. Experiments were performed in triplicates and bars represent mean values for phage TU recovered from each fraction ± SEM. (b) KS1767 cells were incubated with iPhage-NLS for 24 h at 37°C. iPhage-NLS particles were detected in the nucleus by phage antibody. Arrowheads indicate merged red-blue fluorescence. Scale Bar, 100 μm.

Figure 7

Combinatorial screening of peptides targeting subcellular compartments and identification of candidate receptors by affinity chromatography and MS/MS analysis. (a) Diagram of combinatorial screening and peptide validation steps. (b) Selection of an iPhage display peptide library from nuclei, cytosol, and mitochondrial/ER fractions produces serial sequence enrichment. (c) Cellular distribution of internalizing candidate peptide fused to pen depicts peptide homing and binding to the nuclei of KS1767 cells. Intracellular peptide location was revealed by avidin-FITC and DAPI counterstaining. Arrows indicate peptide localization. Scale Bar, 100 μm. (d) Wash, peptide-elution, and glycine elution fractions were immobilized on 96-well plates and tested for binding with iPhage harboring the iLP peptide. Insertless iPhage and BSA were used as negative controls. Phage binding assays were run in triplicates; bars represent mean values for phage TU recovered from immobilized proteins ± SEM. (e) Wash (F22) and elution (F46) fractions were analyzed on a Tris-glycine gel (4% – 20%), and proteins were visualized by Coomassie blue staining. Arrows indicate proteins extracted for mass spectrometric analysis.