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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

Antigen-specific enrichment in affinity selection depends on capsid display scaffold.Two rounds of affinity selection were performed using two different scFv specificities displayed on either of pIII, pVII or pIX. In each library, a total of 12, the specific scFv was spiked into a large background of virions (1∶107) as described in Methods. All selections were done in parallel on both antigens (phOx-BSA and NIP-BSA) employing three different virion elution conditions following antigen binding; high pH (TEA), direct in-well infection, or proteolytic antigen-virion disruption (trypsin). Following amplification, equal volumes of virion-containing culture supernatants from selection round 1 and 2 were assessed for antigen reactivity by phage capture ELISA. Round 0 corresponds to the spiked input of 1×1010 cfuampR. To estimate maximum possible response (100% enrichment of the specific scFv), culture supernatants from the pure specific virions that were spiked into the libraries were used as reference for the corresponding selection. The results are given as fraction of the maximum, indicated by cone shape. Importantly, the virion titers of all supernatants were roughly equal (data not shown).
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pone-0017433-g006: Antigen-specific enrichment in affinity selection depends on capsid display scaffold.Two rounds of affinity selection were performed using two different scFv specificities displayed on either of pIII, pVII or pIX. In each library, a total of 12, the specific scFv was spiked into a large background of virions (1∶107) as described in Methods. All selections were done in parallel on both antigens (phOx-BSA and NIP-BSA) employing three different virion elution conditions following antigen binding; high pH (TEA), direct in-well infection, or proteolytic antigen-virion disruption (trypsin). Following amplification, equal volumes of virion-containing culture supernatants from selection round 1 and 2 were assessed for antigen reactivity by phage capture ELISA. Round 0 corresponds to the spiked input of 1×1010 cfuampR. To estimate maximum possible response (100% enrichment of the specific scFv), culture supernatants from the pure specific virions that were spiked into the libraries were used as reference for the corresponding selection. The results are given as fraction of the maximum, indicated by cone shape. Importantly, the virion titers of all supernatants were roughly equal (data not shown).

Mentions: To elucidate how pVII and pIX display perform in affinity selection, we again compared them with conventional pIII display. Virions were produced in the presence or absence of IPTG induction, the POIs being either scFv anti-phOx or anti-NIP, respectively. Thus, a total of 6 phage populations were evaluated for the two antigens phOx- and NIP-BSA. In each case, the antigen specific virions were mixed with the specificity irrelevant virion at a ratio of 1∶107. The two scFvs do not cross-react (data not shown). Two rounds of affinity selection were then carried out. We used three different elution strategies; high pH, proteolysis with trypsin or direct infection. Enrichment was analysed by comparing the signals from the unselected mock library (R0) with the outputs of the first (R1) and second (R2) round of selection using an antigen specific phage capture ELISA (Fig. 6). The results showed that pVII and pIX perform better than pIII in affinity selection in all but one case, namely phOx selection using elution by direct infection. The standard pIII display route employing high pH (TEA) or proteolytic (trypsin) elution exhibited poor enrichment compared to pVII and pIX. Selection was more efficient without than with IPTG induction, independently of display route and elution conditions, and the negative effect of IPTG induction was most severe for pIII display route.


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)

Antigen-specific enrichment in affinity selection depends on capsid display scaffold.Two rounds of affinity selection were performed using two different scFv specificities displayed on either of pIII, pVII or pIX. In each library, a total of 12, the specific scFv was spiked into a large background of virions (1∶107) as described in Methods. All selections were done in parallel on both antigens (phOx-BSA and NIP-BSA) employing three different virion elution conditions following antigen binding; high pH (TEA), direct in-well infection, or proteolytic antigen-virion disruption (trypsin). Following amplification, equal volumes of virion-containing culture supernatants from selection round 1 and 2 were assessed for antigen reactivity by phage capture ELISA. Round 0 corresponds to the spiked input of 1×1010 cfuampR. To estimate maximum possible response (100% enrichment of the specific scFv), culture supernatants from the pure specific virions that were spiked into the libraries were used as reference for the corresponding selection. The results are given as fraction of the maximum, indicated by cone shape. Importantly, the virion titers of all supernatants were roughly equal (data not shown).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044770&req=5

pone-0017433-g006: Antigen-specific enrichment in affinity selection depends on capsid display scaffold.Two rounds of affinity selection were performed using two different scFv specificities displayed on either of pIII, pVII or pIX. In each library, a total of 12, the specific scFv was spiked into a large background of virions (1∶107) as described in Methods. All selections were done in parallel on both antigens (phOx-BSA and NIP-BSA) employing three different virion elution conditions following antigen binding; high pH (TEA), direct in-well infection, or proteolytic antigen-virion disruption (trypsin). Following amplification, equal volumes of virion-containing culture supernatants from selection round 1 and 2 were assessed for antigen reactivity by phage capture ELISA. Round 0 corresponds to the spiked input of 1×1010 cfuampR. To estimate maximum possible response (100% enrichment of the specific scFv), culture supernatants from the pure specific virions that were spiked into the libraries were used as reference for the corresponding selection. The results are given as fraction of the maximum, indicated by cone shape. Importantly, the virion titers of all supernatants were roughly equal (data not shown).
Mentions: To elucidate how pVII and pIX display perform in affinity selection, we again compared them with conventional pIII display. Virions were produced in the presence or absence of IPTG induction, the POIs being either scFv anti-phOx or anti-NIP, respectively. Thus, a total of 6 phage populations were evaluated for the two antigens phOx- and NIP-BSA. In each case, the antigen specific virions were mixed with the specificity irrelevant virion at a ratio of 1∶107. The two scFvs do not cross-react (data not shown). Two rounds of affinity selection were then carried out. We used three different elution strategies; high pH, proteolysis with trypsin or direct infection. Enrichment was analysed by comparing the signals from the unselected mock library (R0) with the outputs of the first (R1) and second (R2) round of selection using an antigen specific phage capture ELISA (Fig. 6). The results showed that pVII and pIX perform better than pIII in affinity selection in all but one case, namely phOx selection using elution by direct infection. The standard pIII display route employing high pH (TEA) or proteolytic (trypsin) elution exhibited poor enrichment compared to pVII and pIX. Selection was more efficient without than with IPTG induction, independently of display route and elution conditions, and the negative effect of IPTG induction was most severe for pIII display route.

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