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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

Efficiency of the panning procedures. Different phage libraries, C6 and L15, were used in both a tube biopanning and a chromato-panning procedure. In both procedures three rounds were performed as described in Table 1. The binding efficiency of the obtained phages was determined using HuLF ELISA as described in Materials and Methods. (a) Biopanning with a C6 library. Tube panning: Round 1 (black square) Round 3 (open square); column panning: Round 1 (black circle-dotted line). (b) Biopanning with a L15 library. Tube panning: Round 1 (black square) and Round 3 (open square); column panning: Round 1 (black circle-dotted line).
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Figure 2: Efficiency of the panning procedures. Different phage libraries, C6 and L15, were used in both a tube biopanning and a chromato-panning procedure. In both procedures three rounds were performed as described in Table 1. The binding efficiency of the obtained phages was determined using HuLF ELISA as described in Materials and Methods. (a) Biopanning with a C6 library. Tube panning: Round 1 (black square) Round 3 (open square); column panning: Round 1 (black circle-dotted line). (b) Biopanning with a L15 library. Tube panning: Round 1 (black square) and Round 3 (open square); column panning: Round 1 (black circle-dotted line).

Mentions: The efficiency of the column panning procedure (Figure 1b) was studied by comparing the performance of the chromato-panning procedure with that of the tube panning procedure. Different phage libraries were used, and three panning rounds were performed. Human lactoferrin (HuLF) was used as the ligand, and this was coupled to the column as described in Materials and Methods. The most frequently used elution media in biopanning procedures are glycine and hydrochloric acid at a pH of ~2.2. Frequent use of these acidic buffers may denature the bound ligand, leading to a loss in binding capacity. In earlier work [25,26] 1 M NaCl was used as elution medium to elute protein from a cryogel-phage column with good results. To evaluate the elution strength both elution media were investigated in a column panning procedure, showing similar elution efficiencies (Figure 1d). Based on these findings both elution solvents were used in further experiments: (i) for the tube panning procedure, 0.1 M glycine, pH 2.2, was used as the elution medium and (ii) for the column panning procedure 1 M NaCl was used as elution medium. After elution, phages from each panning round were amplified and recovered, their binding efficiencies were tested using a phage ELISA as described in Materials and Methods. Different phage libraries, a cyclic hexamer (C6), linear hexamer (L6) and a linear pentadecamer (L15) library, were used for biopanning. After one round in the column panning procedure, the selected phages show comparable (C6 library) or somewhat higher (L15 library) binding strength towards HuLF as compared to the phages obtained after the third panning round in the tube panning experiment (Figure 2a, b). The L6 library showed similar results to the C6 library (data not shown). In the tube panning experiment an increase in binding strength was observed in consecutive panning rounds (R3 > R2 > R1). In the column panning procedure, no further increase was observed after the first round (L6 and C6 library). Table 1. shows that a statistical significant difference (p < 0.05) between top-values (plateau levels) is observed for all three used between the first round of the tube panning and the chromato-panning procedure, which disappeared after the third tube panning round. Furthermore, also a paired comparison over the three libraries between the first round of the tube panning and the chromato-panning procedure resulted in a statistically significant difference (p = 0.025). These results show the superiority of the chromato-panning procedure for fast screening for high affinity ligands.


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)

Efficiency of the panning procedures. Different phage libraries, C6 and L15, were used in both a tube biopanning and a chromato-panning procedure. In both procedures three rounds were performed as described in Table 1. The binding efficiency of the obtained phages was determined using HuLF ELISA as described in Materials and Methods. (a) Biopanning with a C6 library. Tube panning: Round 1 (black square) Round 3 (open square); column panning: Round 1 (black circle-dotted line). (b) Biopanning with a L15 library. Tube panning: Round 1 (black square) and Round 3 (open square); column panning: Round 1 (black circle-dotted line).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Efficiency of the panning procedures. Different phage libraries, C6 and L15, were used in both a tube biopanning and a chromato-panning procedure. In both procedures three rounds were performed as described in Table 1. The binding efficiency of the obtained phages was determined using HuLF ELISA as described in Materials and Methods. (a) Biopanning with a C6 library. Tube panning: Round 1 (black square) Round 3 (open square); column panning: Round 1 (black circle-dotted line). (b) Biopanning with a L15 library. Tube panning: Round 1 (black square) and Round 3 (open square); column panning: Round 1 (black circle-dotted line).
Mentions: The efficiency of the column panning procedure (Figure 1b) was studied by comparing the performance of the chromato-panning procedure with that of the tube panning procedure. Different phage libraries were used, and three panning rounds were performed. Human lactoferrin (HuLF) was used as the ligand, and this was coupled to the column as described in Materials and Methods. The most frequently used elution media in biopanning procedures are glycine and hydrochloric acid at a pH of ~2.2. Frequent use of these acidic buffers may denature the bound ligand, leading to a loss in binding capacity. In earlier work [25,26] 1 M NaCl was used as elution medium to elute protein from a cryogel-phage column with good results. To evaluate the elution strength both elution media were investigated in a column panning procedure, showing similar elution efficiencies (Figure 1d). Based on these findings both elution solvents were used in further experiments: (i) for the tube panning procedure, 0.1 M glycine, pH 2.2, was used as the elution medium and (ii) for the column panning procedure 1 M NaCl was used as elution medium. After elution, phages from each panning round were amplified and recovered, their binding efficiencies were tested using a phage ELISA as described in Materials and Methods. Different phage libraries, a cyclic hexamer (C6), linear hexamer (L6) and a linear pentadecamer (L15) library, were used for biopanning. After one round in the column panning procedure, the selected phages show comparable (C6 library) or somewhat higher (L15 library) binding strength towards HuLF as compared to the phages obtained after the third panning round in the tube panning experiment (Figure 2a, b). The L6 library showed similar results to the C6 library (data not shown). In the tube panning experiment an increase in binding strength was observed in consecutive panning rounds (R3 > R2 > R1). In the column panning procedure, no further increase was observed after the first round (L6 and C6 library). Table 1. shows that a statistical significant difference (p < 0.05) between top-values (plateau levels) is observed for all three used between the first round of the tube panning and the chromato-panning procedure, which disappeared after the third tube panning round. Furthermore, also a paired comparison over the three libraries between the first round of the tube panning and the chromato-panning procedure resulted in a statistically significant difference (p = 0.025). These results show the superiority of the chromato-panning procedure for fast screening for high affinity ligands.

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