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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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Agarose gel electrophoresis of the RNA display system vector construction.Panel A) Lanes 1–3: positive recombinant pUCHisJ plasmid clone (cl) restricted with AflII and NsiI; Lanes 5–7: positive pUCtHisF and Lane 8: negative clone. Panel B) Lanes 2–6: positive recombinants pBRT7QβHisJ restricted with AflII and NsiI; Lane 7: negative clone. Panel C) Lanes 2–7: positive recombinants pBRT7QβtHisF restricted with AflII and NsiI. Panel D): Lane 1: pQβ8 negative control; Lanes 2 and 3: positive recombinants pQβ8ΔA1; Lanes 4 and 5: positive recombinants pBRT7Qβ-FMDV; Lanes 6 and 7: positive recombinants pBRT7QβΔA1 all restricted with NheI. Lanes “ladder” were loaded with the 100 bp or 1 kb DNA ladder.
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pone-0113069-g003: Agarose gel electrophoresis of the RNA display system vector construction.Panel A) Lanes 1–3: positive recombinant pUCHisJ plasmid clone (cl) restricted with AflII and NsiI; Lanes 5–7: positive pUCtHisF and Lane 8: negative clone. Panel B) Lanes 2–6: positive recombinants pBRT7QβHisJ restricted with AflII and NsiI; Lane 7: negative clone. Panel C) Lanes 2–7: positive recombinants pBRT7QβtHisF restricted with AflII and NsiI. Panel D): Lane 1: pQβ8 negative control; Lanes 2 and 3: positive recombinants pQβ8ΔA1; Lanes 4 and 5: positive recombinants pBRT7Qβ-FMDV; Lanes 6 and 7: positive recombinants pBRT7QβΔA1 all restricted with NheI. Lanes “ladder” were loaded with the 100 bp or 1 kb DNA ladder.

Mentions: Initially, two variants of the pBRT7Qβ plasmid were constructed: pBRT7QβESPI and pBRT7QβNOTI. In these plasmids, additional nucleotides were added to the 3′end of A1 gene to introduce multiple cloning sites (Fig. 1). For pBRT7QβESPI, 6 nucleotides were added to introduce an EspI site, and 9 nucleotides were added to pBRT7QβNOTI to introduce a Not I site. We tested if these extensions allowed proper DNA packing and the production of infectious-competent phage. Indeed, we show that 3 different gene fusions with A1 placed in front of the natural opal and ochre stop codons (TGA and TAA), produced phage plaques in bacterial lawns (Fig. 2). These results suggest that the 3′- end of A1 can accept minor extensions without disturbing the function of phage infectivity. We next explored the lengths of extensions and their effect on infectivity. Various DNA lengths (15–850 bp) were successfully fused with the A1 gene (Figs. 2 & 3), but only recombinant plasmids containing foreign inserted DNA with lengths between 15–300 bp produced phage plaques. These results show that the length of the inserted DNA is critically important for this novel system. Next we tested whether the 3′ end of the A1 gene is critical and important. To accomplish this, we constructed the plasmid pBRT7QβΔA1, in which non-essential sequences of the cDNA of the Qβ genome were deleted from the 3′-terminus of the A1 protein gene. Specifically, we deleted a 162 bp part of the 3′ terminus of the A1 gene (between nucleotides 2271 and 2333) and replaced it with a short adaptor gene sequence of 33 bp leaving the original intercistronic region between A1 gene and the replicase gene intact. Interestingly, these recombinant plasmids with 3′ truncations of A1, still produced phage plaques. However, further deletion of the A1 protein gene beyond nucleotides 2271 at 5′ end or 2333 at 3′ end abolished phage production. Furthermore, we tested whether the orientation of Qβ cDNA within the plasmid is critical. We created identical constructs using pBRT7Qβ and pQβ8, both of which contain the entire cDNA of phage Qβ albeit in opposite orientations. These plasmids yielded phages with similar titers to the wt, suggesting that the orientation of phage cDNA does not influence the phage production. Positive recombinant pBRT7Qβ or pQβ8 plasmids were identified via restriction enzyme (Fig. 3) prior to sequencing and transforming into Qβ for characterizing the display of foreign peptides and proteins.


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

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

Agarose gel electrophoresis of the RNA display system vector construction.Panel A) Lanes 1–3: positive recombinant pUCHisJ plasmid clone (cl) restricted with AflII and NsiI; Lanes 5–7: positive pUCtHisF and Lane 8: negative clone. Panel B) Lanes 2–6: positive recombinants pBRT7QβHisJ restricted with AflII and NsiI; Lane 7: negative clone. Panel C) Lanes 2–7: positive recombinants pBRT7QβtHisF restricted with AflII and NsiI. Panel D): Lane 1: pQβ8 negative control; Lanes 2 and 3: positive recombinants pQβ8ΔA1; Lanes 4 and 5: positive recombinants pBRT7Qβ-FMDV; Lanes 6 and 7: positive recombinants pBRT7QβΔA1 all restricted with NheI. Lanes “ladder” were loaded with the 100 bp or 1 kb DNA ladder.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113069-g003: Agarose gel electrophoresis of the RNA display system vector construction.Panel A) Lanes 1–3: positive recombinant pUCHisJ plasmid clone (cl) restricted with AflII and NsiI; Lanes 5–7: positive pUCtHisF and Lane 8: negative clone. Panel B) Lanes 2–6: positive recombinants pBRT7QβHisJ restricted with AflII and NsiI; Lane 7: negative clone. Panel C) Lanes 2–7: positive recombinants pBRT7QβtHisF restricted with AflII and NsiI. Panel D): Lane 1: pQβ8 negative control; Lanes 2 and 3: positive recombinants pQβ8ΔA1; Lanes 4 and 5: positive recombinants pBRT7Qβ-FMDV; Lanes 6 and 7: positive recombinants pBRT7QβΔA1 all restricted with NheI. Lanes “ladder” were loaded with the 100 bp or 1 kb DNA ladder.
Mentions: Initially, two variants of the pBRT7Qβ plasmid were constructed: pBRT7QβESPI and pBRT7QβNOTI. In these plasmids, additional nucleotides were added to the 3′end of A1 gene to introduce multiple cloning sites (Fig. 1). For pBRT7QβESPI, 6 nucleotides were added to introduce an EspI site, and 9 nucleotides were added to pBRT7QβNOTI to introduce a Not I site. We tested if these extensions allowed proper DNA packing and the production of infectious-competent phage. Indeed, we show that 3 different gene fusions with A1 placed in front of the natural opal and ochre stop codons (TGA and TAA), produced phage plaques in bacterial lawns (Fig. 2). These results suggest that the 3′- end of A1 can accept minor extensions without disturbing the function of phage infectivity. We next explored the lengths of extensions and their effect on infectivity. Various DNA lengths (15–850 bp) were successfully fused with the A1 gene (Figs. 2 & 3), but only recombinant plasmids containing foreign inserted DNA with lengths between 15–300 bp produced phage plaques. These results show that the length of the inserted DNA is critically important for this novel system. Next we tested whether the 3′ end of the A1 gene is critical and important. To accomplish this, we constructed the plasmid pBRT7QβΔA1, in which non-essential sequences of the cDNA of the Qβ genome were deleted from the 3′-terminus of the A1 protein gene. Specifically, we deleted a 162 bp part of the 3′ terminus of the A1 gene (between nucleotides 2271 and 2333) and replaced it with a short adaptor gene sequence of 33 bp leaving the original intercistronic region between A1 gene and the replicase gene intact. Interestingly, these recombinant plasmids with 3′ truncations of A1, still produced phage plaques. However, further deletion of the A1 protein gene beyond nucleotides 2271 at 5′ end or 2333 at 3′ end abolished phage production. Furthermore, we tested whether the orientation of Qβ cDNA within the plasmid is critical. We created identical constructs using pBRT7Qβ and pQβ8, both of which contain the entire cDNA of phage Qβ albeit in opposite orientations. These plasmids yielded phages with similar titers to the wt, suggesting that the orientation of phage cDNA does not influence the phage production. Positive recombinant pBRT7Qβ or pQβ8 plasmids were identified via restriction enzyme (Fig. 3) prior to sequencing and transforming into Qβ for characterizing the display of foreign peptides and proteins.

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