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Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries.

Noppe W, Plieva F, Galaev IY, Pottel H, Deckmyn H, Mattiasson B - BMC Biotechnol. (2009)

Bottom Line: Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel.After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out.Chromato-panning allows combining several steps of the panning procedure resulting in 4-8 fold decrease of total time needed for phage selection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Lund University, Lund, Sweden. Wim.Noppe@kuleuven-kortrijk.be

ABSTRACT

Background: Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library.

Results: Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. E. coli cells were applied on the column for infection with the specifically bound phages.

Conclusion: Chromato-panning allows combining several steps of the panning procedure resulting in 4-8 fold decrease of total time needed for phage selection.

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Related in: MedlinePlus

On-column E. coli infection. Two phage libraries, L6 and L15, were used for column panning and on-column infection. Two rounds were performed as described in Table 1. As a reference sample, a single phage clone HuN, was used for on-column infection. The binding efficiency was determined using HuLF ELISA as described in Material and Methods. (a) On-column cell infection on a HuLF column with the HuN single-phage clone. HuN phage reference sample (no infection performed) (black square) and HuN fraction eluted with 1 M NaCl after on-column infection (open square-dotted line). (b) Column panning and on-column cell infection on the HuLF column with the L6 and L15 phage peptide libraries. Two panning rounds were performed. Phages from the L6 phage peptide library (solid lines): Round 1 (black square) and Round 2 (open square); phages from the L15 phage peptide library (dotted lines): Round 1 (black circle) and Round 2 (open circle ○). (c) Control of cell growth on agar plates after on-column phage infection. (left): E. coli cells which were not infected with phages were plated on agar plates containing tetracycline and incubated overnight at 37°C. (right): E. coli cells were applied to the HuLF column on which phages were bound after a panning round. After cell infection and elution, the 1 M NaCl fraction was plated on agar plates containing tetracycline and grown overnight at 37°C.
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Figure 3: On-column E. coli infection. Two phage libraries, L6 and L15, were used for column panning and on-column infection. Two rounds were performed as described in Table 1. As a reference sample, a single phage clone HuN, was used for on-column infection. The binding efficiency was determined using HuLF ELISA as described in Material and Methods. (a) On-column cell infection on a HuLF column with the HuN single-phage clone. HuN phage reference sample (no infection performed) (black square) and HuN fraction eluted with 1 M NaCl after on-column infection (open square-dotted line). (b) Column panning and on-column cell infection on the HuLF column with the L6 and L15 phage peptide libraries. Two panning rounds were performed. Phages from the L6 phage peptide library (solid lines): Round 1 (black square) and Round 2 (open square); phages from the L15 phage peptide library (dotted lines): Round 1 (black circle) and Round 2 (open circle ○). (c) Control of cell growth on agar plates after on-column phage infection. (left): E. coli cells which were not infected with phages were plated on agar plates containing tetracycline and incubated overnight at 37°C. (right): E. coli cells were applied to the HuLF column on which phages were bound after a panning round. After cell infection and elution, the 1 M NaCl fraction was plated on agar plates containing tetracycline and grown overnight at 37°C.

Mentions: The on-column infection procedure was first tested with a single-clone phage (HuN phage clone) as described in Materials and Methods. The amount of amplified phages after on-column infection was comparable to the amount obtained in a tube panning procedure or a column panning procedure with a separate infection step. The binding strength of the phages obtained was similar to that for phages obtained from a normal panning procedure (Figure 3a). A similar result was obtained with phage clones Hu14 and Hu5 (data not shown) although the three phage clones expressed a different peptide sequence on the pIII protein [7]. The same procedure was consequently performed with the L6 and L15 phage libraries. Two column panning rounds and on-column infection were performed with each phage library. The amount of phages and the binding strength were determined after each panning round. Again, the amount of phages was comparable to the amount of phages obtained in the other panning procedures. The binding strength was also similar as that obtained in the column panning procedure with a separate infection step. Again, the results show that only one panning round is necessary to obtain specific phages for the L6 library. Statistical analysis shows no significant difference between the top-values of round 1 and 2 of the chromato-panning (95% CI round 1: 1.069–1.432; 95% CI round 2: 1.199–1.393). For the L15 library still some increase is observed after the second round (95% CI round 1: 1.295–1.550; 95% CI round 2: 1.627–2.176), probably due to the higher diversity of phages present in the library (Figure 3b), indicating that a second panning round might here be advisable. These results are in agreement with those shown in Figure 2b, where a similar column panning procedure was performed except that the infection step was performed separately and not on-column. These results confirm that biopanning was achieved on the HuLF column, and that on-column infection of the E. coli cells took place.


Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries.

Noppe W, Plieva F, Galaev IY, Pottel H, Deckmyn H, Mattiasson B - BMC Biotechnol. (2009)

On-column E. coli infection. Two phage libraries, L6 and L15, were used for column panning and on-column infection. Two rounds were performed as described in Table 1. As a reference sample, a single phage clone HuN, was used for on-column infection. The binding efficiency was determined using HuLF ELISA as described in Material and Methods. (a) On-column cell infection on a HuLF column with the HuN single-phage clone. HuN phage reference sample (no infection performed) (black square) and HuN fraction eluted with 1 M NaCl after on-column infection (open square-dotted line). (b) Column panning and on-column cell infection on the HuLF column with the L6 and L15 phage peptide libraries. Two panning rounds were performed. Phages from the L6 phage peptide library (solid lines): Round 1 (black square) and Round 2 (open square); phages from the L15 phage peptide library (dotted lines): Round 1 (black circle) and Round 2 (open circle ○). (c) Control of cell growth on agar plates after on-column phage infection. (left): E. coli cells which were not infected with phages were plated on agar plates containing tetracycline and incubated overnight at 37°C. (right): E. coli cells were applied to the HuLF column on which phages were bound after a panning round. After cell infection and elution, the 1 M NaCl fraction was plated on agar plates containing tetracycline and grown overnight at 37°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667499&req=5

Figure 3: On-column E. coli infection. Two phage libraries, L6 and L15, were used for column panning and on-column infection. Two rounds were performed as described in Table 1. As a reference sample, a single phage clone HuN, was used for on-column infection. The binding efficiency was determined using HuLF ELISA as described in Material and Methods. (a) On-column cell infection on a HuLF column with the HuN single-phage clone. HuN phage reference sample (no infection performed) (black square) and HuN fraction eluted with 1 M NaCl after on-column infection (open square-dotted line). (b) Column panning and on-column cell infection on the HuLF column with the L6 and L15 phage peptide libraries. Two panning rounds were performed. Phages from the L6 phage peptide library (solid lines): Round 1 (black square) and Round 2 (open square); phages from the L15 phage peptide library (dotted lines): Round 1 (black circle) and Round 2 (open circle ○). (c) Control of cell growth on agar plates after on-column phage infection. (left): E. coli cells which were not infected with phages were plated on agar plates containing tetracycline and incubated overnight at 37°C. (right): E. coli cells were applied to the HuLF column on which phages were bound after a panning round. After cell infection and elution, the 1 M NaCl fraction was plated on agar plates containing tetracycline and grown overnight at 37°C.
Mentions: The on-column infection procedure was first tested with a single-clone phage (HuN phage clone) as described in Materials and Methods. The amount of amplified phages after on-column infection was comparable to the amount obtained in a tube panning procedure or a column panning procedure with a separate infection step. The binding strength of the phages obtained was similar to that for phages obtained from a normal panning procedure (Figure 3a). A similar result was obtained with phage clones Hu14 and Hu5 (data not shown) although the three phage clones expressed a different peptide sequence on the pIII protein [7]. The same procedure was consequently performed with the L6 and L15 phage libraries. Two column panning rounds and on-column infection were performed with each phage library. The amount of phages and the binding strength were determined after each panning round. Again, the amount of phages was comparable to the amount of phages obtained in the other panning procedures. The binding strength was also similar as that obtained in the column panning procedure with a separate infection step. Again, the results show that only one panning round is necessary to obtain specific phages for the L6 library. Statistical analysis shows no significant difference between the top-values of round 1 and 2 of the chromato-panning (95% CI round 1: 1.069–1.432; 95% CI round 2: 1.199–1.393). For the L15 library still some increase is observed after the second round (95% CI round 1: 1.295–1.550; 95% CI round 2: 1.627–2.176), probably due to the higher diversity of phages present in the library (Figure 3b), indicating that a second panning round might here be advisable. These results are in agreement with those shown in Figure 2b, where a similar column panning procedure was performed except that the infection step was performed separately and not on-column. These results confirm that biopanning was achieved on the HuLF column, and that on-column infection of the E. coli cells took place.

Bottom Line: Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel.After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out.Chromato-panning allows combining several steps of the panning procedure resulting in 4-8 fold decrease of total time needed for phage selection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Lund University, Lund, Sweden. Wim.Noppe@kuleuven-kortrijk.be

ABSTRACT

Background: Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library.

Results: Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. E. coli cells were applied on the column for infection with the specifically bound phages.

Conclusion: Chromato-panning allows combining several steps of the panning procedure resulting in 4-8 fold decrease of total time needed for phage selection.

Show MeSH
Related in: MedlinePlus