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In vitro evolution and affinity-maturation with Coliphage qβ display.

Skamel C, Aller SG, Bopda Waffo A - PLoS ONE (2014)

Bottom Line: DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide.The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy.Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

View Article: PubMed Central - PubMed

Affiliation: Campus Technologies Freiburg (CTF) GmbH, Agency for Technology Transfer at the University and University Medical Center Freiburg, Freiburg, Germany.

ABSTRACT
The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

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Related in: MedlinePlus

Sequences comparison of the randomized FMDV G-H loop displayed after three rounds of Biopanning.The first line up is the original loop motif Arg-Gly-Asp of VP1 G-H loop of FMDV strain S8C. On the left hand side under the original sequence are the sequences obtained after a round of biopanning. The low case letters show the different between the sequences. The last line is the expression of the evolution of the original motif sequence on the 3rd round shown the maintanance of Arg-Gly and change of the last amino acid.
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pone-0113069-g008: Sequences comparison of the randomized FMDV G-H loop displayed after three rounds of Biopanning.The first line up is the original loop motif Arg-Gly-Asp of VP1 G-H loop of FMDV strain S8C. On the left hand side under the original sequence are the sequences obtained after a round of biopanning. The low case letters show the different between the sequences. The last line is the expression of the evolution of the original motif sequence on the 3rd round shown the maintanance of Arg-Gly and change of the last amino acid.

Mentions: To gain fitness, the synthesized library of the G-H loop was selected using a modified biopanning protocol [1], [2], [28] with mAb SD6. This modified protocol selects and amplifies phage while avoiding acidic elution of phages selected (Fig. 7). We reasoned that removal of an acidic elution step would enhance phage viability and the overall efficiency of in vitro evolution. Additionally, media containing the indicator bacteria E. coli Q13 grown to the log phase was added directly to the plate to further enhance survival of hybrid phages. After each round, an aliquot of phage was used for RT-PCR and the resulting DNA sequence was compared to the wild type sequence (Fig. 8). The sequence comparison of the randomized VP1 G-H loop after six rounds of biopanning revealed a shift of mAb SD6 binding motif from Arg-Gly-Asp to Xxx-Arg-Gly. Due to the preservation of the Arg and Gly in all three rounds of biopanning, we conclude that this tandem pair is essential for mAb SD6 binding. The third amino acid was substituted without disturbing the binding capacity of the peptide to the mAb SD6. Over 80% of glycine exposed by the phage was in contact with the mAb SD6. This clearly shows that arginine and glycine were not just only together in the antibody binding motif but were representing optimized amino acid from a randomized pool.


In vitro evolution and affinity-maturation with Coliphage qβ display.

Skamel C, Aller SG, Bopda Waffo A - PLoS ONE (2014)

Sequences comparison of the randomized FMDV G-H loop displayed after three rounds of Biopanning.The first line up is the original loop motif Arg-Gly-Asp of VP1 G-H loop of FMDV strain S8C. On the left hand side under the original sequence are the sequences obtained after a round of biopanning. The low case letters show the different between the sequences. The last line is the expression of the evolution of the original motif sequence on the 3rd round shown the maintanance of Arg-Gly and change of the last amino acid.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231119&req=5

pone-0113069-g008: Sequences comparison of the randomized FMDV G-H loop displayed after three rounds of Biopanning.The first line up is the original loop motif Arg-Gly-Asp of VP1 G-H loop of FMDV strain S8C. On the left hand side under the original sequence are the sequences obtained after a round of biopanning. The low case letters show the different between the sequences. The last line is the expression of the evolution of the original motif sequence on the 3rd round shown the maintanance of Arg-Gly and change of the last amino acid.
Mentions: To gain fitness, the synthesized library of the G-H loop was selected using a modified biopanning protocol [1], [2], [28] with mAb SD6. This modified protocol selects and amplifies phage while avoiding acidic elution of phages selected (Fig. 7). We reasoned that removal of an acidic elution step would enhance phage viability and the overall efficiency of in vitro evolution. Additionally, media containing the indicator bacteria E. coli Q13 grown to the log phase was added directly to the plate to further enhance survival of hybrid phages. After each round, an aliquot of phage was used for RT-PCR and the resulting DNA sequence was compared to the wild type sequence (Fig. 8). The sequence comparison of the randomized VP1 G-H loop after six rounds of biopanning revealed a shift of mAb SD6 binding motif from Arg-Gly-Asp to Xxx-Arg-Gly. Due to the preservation of the Arg and Gly in all three rounds of biopanning, we conclude that this tandem pair is essential for mAb SD6 binding. The third amino acid was substituted without disturbing the binding capacity of the peptide to the mAb SD6. Over 80% of glycine exposed by the phage was in contact with the mAb SD6. This clearly shows that arginine and glycine were not just only together in the antibody binding motif but were representing optimized amino acid from a randomized pool.

Bottom Line: DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide.The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy.Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

View Article: PubMed Central - PubMed

Affiliation: Campus Technologies Freiburg (CTF) GmbH, Agency for Technology Transfer at the University and University Medical Center Freiburg, Freiburg, Germany.

ABSTRACT
The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

Show MeSH
Related in: MedlinePlus