Phage display technology is being increasingly used in translational research as a tool for validating novel therapeutic targets and drug design. It has multiple uses in studying for example, protein-protein interactions, enzyme specificity, antibody-antigen interaction and epitope mapping231. The principle is centred on the construction of a phage library, approximately 3x1010 in size, consisting usually of random peptides. Insertion of a foreign DNA fragment into the coat protein gene of a phagemid vector causes fusion of the peptide with the N- terminus of the coat protein and is thus expressed externally on the phage particle. The genetic information encoding the peptide is held within the phage which can be sequenced and therefore connects genotype and phenotype. Incubation of the phage library with a target molecule selects the phage that bind which can be sequenced to identify the peptide residues involved. This is achieved by a process called biopanning which involves immobilising a target molecule to a 96 well microtiter plate and adding the phage library to the wells. Unbound phage are removed by washing steps followed by elution of the target
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bound phage which are subsequently amplified and a second library (containing increasingly specific phage) produced ready for a second round of biopanning. This process is repeated around 3-6 times, after which the phage particles can be anaylsed by DNA sequencing.
Figure 2-5 Illustration of steps involved in phage display screening
The protein of interest is incubated with the phage library and the unbound phage is washed off. The bound phage is eluted and amplified within E.coli cells and the addition of helper phage. A second phage library is produced and the biopanning process is repeated.
Two separate phage display libraries were used during my research to screen for binders to C3 and fibrinogen and the production of these will be described in more detail in Chapters 4 and 5. For both libraries the peptide sequences were inserted into the coat protein 3 gene (pIII) within a phagemid vector. A phagemid is similar to a plasmid but also contains the f1 phage origin of replication. The vector was electroporated into electrocompetent E.Coli cells containing an F’ pilus. In order for single stranded DNA replication to occur, it is
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necessary to superinfect the E.coli cells with helper phage (e.g. VCSM13 or M13K07), which contain the necessary genes for phage assembly. The advantage of infection of a bacterial host cell with a phagemid and helper phage in this manner allows the display of larger peptides with fewer copies of the coat protein.
2.9.1 Fibrinogen α chain cDNA phage library
A phage library consisting of fragments of the α chain of fibrinogen was constructed (as detailed in chapter 4) to identify interaction sites between the α chain and C3.
2.9.1.1 Biopanning and enrichment
Commercial C3 (MyBioSource, CA, USA) was immobilised on a 96-well micro- titre plate and left overnight at 4oC. Following removal of the coating buffer the well was blocked by the addition of 400µl of 3% (w/v) BSA for 1 hour followed by washing with TBS/0.5% Tween three times. After suitable washing, 70µl of phage was added and incubated at room temperature for 1 hour, followed by 5 cycles of washing as above in order to remove any unbound phage. To elute the bound phage, 30µl of glycine (pH 2.2) was added and the whole mixture transferred to a clean eppendorf tube along with 6µl unadjusted 2M Tris to neutralise the glycine. To this mixture, 1ml of prepared E.coli cells, XL1-Blue MRF’ (Stratagene, La Jolla, CA, USA) was added and incubated at room temperature for 15minutes. In order to estimate the number of phage particles eluted from round 1 of panning, 10µl was plated on agar containing ampicillin. To generate a new, enriched cDNA phage library for a second round of biopanning, the infected E.coli cells were superinfected with 1012 plaque forming units of VCSM13 (Stratagene) helper phage at room temperature for 30
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minutes. The culture was transferred to 100ml of LB medium containing 50ug/ml ampicillin, 10ug/ml tetracycline and 10ug/ml kanamycin and incubated at 37oC overnight. The phage was precipitated by centrifuging the culture followed by the addition of 0.2 volumes of 40% polyethylene glycol 4000/2.5M NaCl. It was then resuspended in 2-3ml of PBS. In total 4 rounds of panning were performed after which the phage DNA was extracted using a Wizard Minipreps DNA purification system (Promega, Southampton, UK) and sent for sequencing.
2.9.2 Non-antibody scaffold protein (Adhiron) phage library
Collaborators from the BioScreening Technology group at the University of Leeds have developed a phage library containing a non-antibody scaffold protein, termed Adhiron. The protein scaffold is based on a consensus sequence of plant-derived phytocystatins, a cysteine protease inhibitor. The inhibitory sequences within two loops of the protein have been replaced with nine randomised amino acid positions in each loop, producing a library size or 1.3 x 1010 clones. The presence of the peptides within the scaffold maintains the conformation of the peptide which is an important consideration when investigating protein-protein interactions.
2.9.2.1 Biopanning and enrichment
The Adhiron library was used to identify binders to both fibrinogen and C3 and the biopanning was similar to that employed with the fibrinogen α chain library. There was, however, a difference in the method used to immobilise the target protein. Commercial fibrinogen (Calbiochem) or C3 (MyBioSource) were initially biotinylated and added to streptavidin coated wells for 1 hour. The Adhiron phage library was incubated with fibrinogen or C3 for 2.5 hours, after which the
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Figure 2-6 Crystal structure of Adhiron (PDB code 46NT)
The crystal structure of an Adhiron scaffold protein with the loop regions containing the random sequence of 9 amino acids shown in red.
wells were washed 10 times and the bound phage were eluted by the addition of glycine and used to infect 1ml of E. coli ER2738 electrocompetent cells (Lucigen, Wisconsin ,USA). Colonies of the ER2738 cells were grown on LB agar plates overnight followed by inoculation with M13K07 helper phage in order to generate a new library for the second round of panning as described in 2.9.1.1. In total, five rounds of panning were completed, after which individual ER2738 colonies were selected for DNA sequencing.