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Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage.

Løset GÅ, Roos N, Bogen B, Sandlie I - PLoS ONE (2011)

Bottom Line: Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform.In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX.This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immune Regulation, University of Oslo, Oslo, Norway. g.a.loset@imbv.uio.no

ABSTRACT

Background: Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display.

Methodology/principal findings: Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient.

Conclusions/significance: Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before.

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Related in: MedlinePlus

Schematic drawing of the filamentous phage structure.(A) The wt virion is made up of five structural proteins that coat a single stranded DNA genome of about 6.4 kb. (B) In the wt phage there is about 2,700 copies of pVIII and approximately 3–5 copies each of the four proteins pIII, pVI, pVII and pIX, which are found at each tip of the virion [4], [35]. The virion size depends on the genome size at approx. 2.3 nucleotides per pVIII, and hence the length of the particle changes as a function of genome length [36]. The theoretical MW of the mature capsid proteins were calculated from the sequence of VCSM13 (GenBank accession no.: AY598820).
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pone-0017433-g001: Schematic drawing of the filamentous phage structure.(A) The wt virion is made up of five structural proteins that coat a single stranded DNA genome of about 6.4 kb. (B) In the wt phage there is about 2,700 copies of pVIII and approximately 3–5 copies each of the four proteins pIII, pVI, pVII and pIX, which are found at each tip of the virion [4], [35]. The virion size depends on the genome size at approx. 2.3 nucleotides per pVIII, and hence the length of the particle changes as a function of genome length [36]. The theoretical MW of the mature capsid proteins were calculated from the sequence of VCSM13 (GenBank accession no.: AY598820).

Mentions: The wt filamentous phage virions of M13, fd and f1 are composed of a small genome surrounded by a cylinder of coat proteins that measures about 1 µm in length and 8–10 nm in diameter, and has a total of about 2,700 copies of the major coat protein pVIII. In addition, the virion harbors approximately 3–5 copies of each of pIII, pVI, pVII and pIX; pIII and pVI on one virion tip and pVII and pIX on the other (Fig. 1) [4]. pIII is of particular importance since it is the critical component for the early events of E. coli host entry [5]. All are integral E. coli inner membrane proteins before virion assembly [6], but only pIII and pVIII are synthesized as precursors containing classical N-terminal signal sequences [4]. pVII and pIX appear to be synthesized without such signal peptides and consequently do not undergo post-translational processing [4].


Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage.

Løset GÅ, Roos N, Bogen B, Sandlie I - PLoS ONE (2011)

Schematic drawing of the filamentous phage structure.(A) The wt virion is made up of five structural proteins that coat a single stranded DNA genome of about 6.4 kb. (B) In the wt phage there is about 2,700 copies of pVIII and approximately 3–5 copies each of the four proteins pIII, pVI, pVII and pIX, which are found at each tip of the virion [4], [35]. The virion size depends on the genome size at approx. 2.3 nucleotides per pVIII, and hence the length of the particle changes as a function of genome length [36]. The theoretical MW of the mature capsid proteins were calculated from the sequence of VCSM13 (GenBank accession no.: AY598820).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044770&req=5

pone-0017433-g001: Schematic drawing of the filamentous phage structure.(A) The wt virion is made up of five structural proteins that coat a single stranded DNA genome of about 6.4 kb. (B) In the wt phage there is about 2,700 copies of pVIII and approximately 3–5 copies each of the four proteins pIII, pVI, pVII and pIX, which are found at each tip of the virion [4], [35]. The virion size depends on the genome size at approx. 2.3 nucleotides per pVIII, and hence the length of the particle changes as a function of genome length [36]. The theoretical MW of the mature capsid proteins were calculated from the sequence of VCSM13 (GenBank accession no.: AY598820).
Mentions: The wt filamentous phage virions of M13, fd and f1 are composed of a small genome surrounded by a cylinder of coat proteins that measures about 1 µm in length and 8–10 nm in diameter, and has a total of about 2,700 copies of the major coat protein pVIII. In addition, the virion harbors approximately 3–5 copies of each of pIII, pVI, pVII and pIX; pIII and pVI on one virion tip and pVII and pIX on the other (Fig. 1) [4]. pIII is of particular importance since it is the critical component for the early events of E. coli host entry [5]. All are integral E. coli inner membrane proteins before virion assembly [6], but only pIII and pVIII are synthesized as precursors containing classical N-terminal signal sequences [4]. pVII and pIX appear to be synthesized without such signal peptides and consequently do not undergo post-translational processing [4].

Bottom Line: Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform.In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX.This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immune Regulation, University of Oslo, Oslo, Norway. g.a.loset@imbv.uio.no

ABSTRACT

Background: Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display.

Methodology/principal findings: Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient.

Conclusions/significance: Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before.

Show MeSH
Related in: MedlinePlus