Editing Phage display (section)

=== Filamentous phages ===

==== pIII ====

pIII is the protein that determines the infectivity of the virion. pIII is composed of three domains (N1, N2 and CT) connected by glycine-rich linkers.<ref name="Lowman_Clackson_2004">{{cite book |vauthors=Lowman HB, Clackson T | title = Phage display: a practical approach | publisher = Oxford University Press | location = Oxford [Oxfordshire] | year = 2004 | pages = 10–11 | isbn = 978-0-19-963873-4 | chapter =  1.3 }}</ref> The N2 domain binds to the F pilus during virion infection freeing the N1 domain which then interacts with a TolA protein on the surface of the bacterium.<ref name="Lowman_Clackson_2004"/> Insertions within this protein are usually added in position 249 (within a linker region between CT and N2), position 198 (within the N2 domain) and at the [[N-terminus]] (inserted between the N-terminal secretion sequence and the N-terminus of pIII).<ref name="Lowman_Clackson_2004"/> However, when using the BamHI site located at position 198 one must be careful of the unpaired Cysteine residue (C201) that could cause problems during phage display if one is using a non-truncated version of pIII.<ref name="Lowman_Clackson_2004"/>

An advantage of using pIII rather than pVIII is that pIII allows for monovalent display when using a phagemid (plasmid derived from [[Ff phages]]) combined with a helper phage. Moreover, pIII allows for the insertion of larger protein sequences (>100 amino acids)<ref name="pmid10669603">{{cite journal |vauthors=Sidhu SS, Weiss GA, Wells JA | title = High copy display of large proteins on phage for functional selections | journal = J. Mol. Biol. | volume = 296 | issue = 2 | pages = 487–95 |date=February 2000 | pmid = 10669603 | doi = 10.1006/jmbi.1999.3465 }}</ref> and is more tolerant to it than pVIII. However, using pIII as the fusion partner can lead to a decrease in phage infectivity leading to problems such as selection bias caused by difference in phage growth rate<ref name="pmid20583018">{{cite journal | vauthors = Derda R, Tang SK, Whitesides GM | title = Uniform amplification of phage with different growth characteristics in individual compartments consisting of monodisperse droplets | journal = Angew. Chem. Int. Ed. Engl. | volume = 49 | issue = 31 | pages = 5301–4 | date = July 2010 | pmid = 20583018 | pmc = 2963104 | doi = 10.1002/anie.201001143 }}</ref> or even worse, the phage's inability to infect its host.<ref name="Lowman_Clackson_2004"/> Loss of phage infectivity can be avoided by using a phagemid plasmid and a helper phage so that the resultant phage contains both wild type and fusion pIII.<ref name="Lowman_Clackson_2004"/>

cDNA has also been analyzed using pIII via a two complementary leucine zippers system,<ref name="pmid7957259">{{cite journal |vauthors=Crameri R, Jaussi R, Menz G, Blaser K | title = Display of expression products of cDNA libraries on phage surfaces. A versatile screening system for selective isolation of genes by specific gene-product/ligand interaction | journal = Eur. J. Biochem. | volume = 226 | issue = 1 | pages = 53–8 |date=November 1994 | pmid = 7957259 | doi = 10.1111/j.1432-1033.1994.00t53.x }}</ref> Direct Interaction Rescue<ref name="pmid7838733">{{cite journal |vauthors=Gramatikoff K, Georgiev O, Schaffner W | title = Direct interaction rescue, a novel filamentous phage technique to study protein-protein interactions | journal = Nucleic Acids Res. | volume = 22 | issue = 25 | pages = 5761–2 |date=December 1994 | pmid = 7838733 | pmc = 310144 | doi = 10.1093/nar/22.25.5761}}</ref> or by adding an 8-10 amino acid linker between the cDNA and pIII at the C-terminus.<ref name="pmid11034335">{{cite journal |vauthors=Fuh G, Sidhu SS | title = Efficient phage display of polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein | journal = FEBS Lett. | volume = 480 | issue = 2–3 | pages = 231–4 |date=September 2000 | pmid = 11034335 | doi = 10.1016/s0014-5793(00)01946-3| s2cid = 23009887 | doi-access = free | bibcode = 2000FEBSL.480..231F }}</ref>

==== pVIII ====

pVIII is the main coat protein of Ff phages. Peptides are usually fused to the N-terminus of pVIII.<ref name="Lowman_Clackson_2004"/> Usually peptides that can be fused to pVIII are 6-8 amino acids long.<ref name="Lowman_Clackson_2004"/> The size restriction seems to have less to do with structural impediment caused by the added section<ref name="Malik_1998">{{cite journal |vauthors=Malik P, Terry TD, Bellintani F, Perham RN | title = Factors limiting display of foreign peptides on the major coat protein of filamentous bacteriophage capsids and a potential role for leader peptidase | journal = FEBS Lett. | volume = 436 | issue = 2 | pages = 263–6 |date=October 1998 | pmid = 9781692 | doi = 10.1016/s0014-5793(98)01140-5| s2cid = 19331069 | doi-access = free | bibcode = 1998FEBSL.436..263M }}</ref> and more to do with the size exclusion caused by pIV during coat protein export.<ref name="Malik_1998"/> Since there are around 2700 copies of the protein on a typical phages, it is more likely that the protein of interest will be expressed polyvalently even if a phagemid is used.<ref name="Lowman_Clackson_2004"/> This makes the use of this protein unfavorable for the discovery of high affinity binding partners.<ref name="Lowman_Clackson_2004"/>

To overcome the size problem of pVIII, artificial coat proteins have been designed.<ref name="Weiss_Sidhu_2000">{{cite journal |vauthors=Weiss GA, Sidhu SS | title = Design and evolution of artificial M13 coat proteins | journal = J. Mol. Biol. | volume = 300 | issue = 1 | pages = 213–9 |date=June 2000 | pmid = 10864510 | doi = 10.1006/jmbi.2000.3845 }}</ref> An example is Weiss and Sidhu's inverted artificial coat protein (ACP) which allows the display of large proteins at the C-terminus.<ref name="Weiss_Sidhu_2000"/> The ACP's could display a protein of 20kDa, however, only at low levels (mostly only monovalently).<ref name="Weiss_Sidhu_2000"/>

==== pVI ====

pVI has been widely used for the display of cDNA libraries.<ref name="Lowman_Clackson_2004"/> The display of cDNA libraries via phage display is an attractive alternative to the yeast-2-hybrid method for the discovery of interacting proteins and peptides due to its high throughput capability.<ref name="Lowman_Clackson_2004"/> pVI has been used preferentially to pVIII and pIII for the expression of cDNA libraries because one can add the protein of interest to the C-terminus of pVI without greatly affecting pVI's role in phage assembly. This means that the stop codon in the cDNA is no longer an issue.<ref name="pmid9634780">{{cite journal |vauthors=Jespers LS, Messens JH, De Keyser A, Eeckhout D, Van den Brande I, Gansemans YG, Lauwereys MJ, Vlasuk GP, Stanssens PE | title = Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene VI | journal = Bio/Technology | volume = 13 | issue = 4 | pages = 378–82 |date=April 1995 | pmid = 9634780 | doi = 10.1038/nbt0495-378| s2cid = 6171262 }}</ref> However, phage display of cDNA is always limited by the inability of most prokaryotes in producing post-translational modifications present in eukaryotic cells or by the misfolding of multi-domain proteins.

While pVI has been useful for the analysis of cDNA libraries, pIII and pVIII remain the most utilized coat proteins for phage display.<ref name="Lowman_Clackson_2004"/>

==== pVII and pIX ====

In an experiment in 1995, display of Glutathione S-transferase was attempted on both pVII and pIX and failed.<ref name="pmid7616570">{{cite journal |vauthors=Endemann H, Model P | title = Location of filamentous phage minor coat proteins in phage and in infected cells | journal = J. Mol. Biol. | volume = 250 | issue = 4 | pages = 496–506 |date=July 1995 | pmid = 7616570 | doi =  10.1006/jmbi.1995.0393}}</ref> However, phage display of this protein was completed successfully after the addition of a periplasmic signal sequence (pelB or ompA) on the N-terminus.<ref name="pmid10339535">{{cite journal |vauthors=Gao C, Mao S, Lo CH, Wirsching P, Lerner RA, Janda KD | title = Making artificial antibodies: a format for phage display of combinatorial heterodimeric arrays | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 96 | issue = 11 | pages = 6025–30 |date=May 1999 | pmid = 10339535 | pmc = 26829 | doi =  10.1073/pnas.96.11.6025|bibcode = 1999PNAS...96.6025G | doi-access = free }}</ref> In a recent study, it has been shown that AviTag, FLAG and His could be displayed on pVII without the need of a signal sequence.  Then the expression of single chain Fv's (scFv), and single chain T cell receptors (scTCR) were expressed both with and without the signal sequence.<ref name="Løset_2011">{{cite journal |vauthors=Løset GÅ, Roos N, Bogen B, Sandlie I | title = Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage | journal = PLOS ONE | volume = 6 | issue = 2 | pages = e17433 | year = 2011 | pmid = 21390283 | pmc = 3044770 | doi = 10.1371/journal.pone.0017433 |bibcode = 2011PLoSO...617433L | doi-access = free }}</ref>

PelB (an amino acid signal sequence that targets the protein to the periplasm where a signal peptidase then cleaves off PelB) improved the phage display level when compared to pVII and pIX fusions without the signal sequence. However, this led to the incorporation of more helper phage genomes rather than phagemid genomes. In all cases, phage display levels were lower than using pIII fusion. However, lower display might be more favorable for the selection of binders due to lower display being closer to true monovalent display. In five out of six occasions, pVII and pIX fusions without pelB was more efficient than pIII fusions in affinity selection assays. The paper even goes on to state that pVII and pIX display platforms may outperform pIII in the long run.<ref name="Løset_2011"/>

The use of pVII and pIX instead of pIII might also be an advantage because virion rescue may be undertaken without breaking the virion-antigen bond if the pIII used is wild type. Instead, one could cleave in a section between the bead and the antigen to elute. Since the pIII is intact it does not matter whether the antigen remains bound to the phage.<ref name="Løset_2011"/>