Limits...
Novel avian single-chain fragment variable (scFv) targets dietary gluten and related natural grain prolamins, toxic entities of celiac disease.

Stadlmann V, Harant H, Korschineck I, Hermann M, Forster F, Missbichler A - BMC Biotechnol. (2015)

Bottom Line: Similarly, the pseudo-grain amaranth, used as gluten-free alternative, is not targeted by scFv.This data indicate that scFv specifically recognizes toxic cereal peptides relevant in CD.ScFv can be of benefit for future CD treatment regimes.

View Article: PubMed Central - PubMed

Affiliation: Sciotec Diagnostics Technologies GmbH, Tulln, Austria. valeriestadlmann@hotmail.com.

ABSTRACT

Background: Celiac disease (CD) is a chronic, small intestinal inflammatory disease mediated by dietary gluten and related prolamins. The only current therapeutic option is maintenance of a strict life-long gluten-free diet, which implies substantial burden for CD patients. Different treatment regimes might be feasible, including masking of toxic celiac peptides with blocking antibodies or fragments thereof. The objective of this study was therefore to select and produce a recombinant avian single-chain fragment variable (scFv) directed against peptic-tryptic digested gliadin (PT-Gliadin) and related celiac toxic entities.

Results: Gluten-free raised chicken of same age were immunized with PT-Gliadin. Chicken splenic lymphocytes, selected with antigen-coated magnetic beads, served as RNA source for the generation of cDNA. Chicken VH and VL genes were amplified from the cDNA by PCR to generate full-length scFv constructs consisting of VH and VL fragments joined by a linker sequence. ScFv constructs were ligated in a prokaryotic expression vector, which provides a C-terminal hexahistidine tag. ScFvs from several bacterial clones were expressed in soluble form and crude cell lysates screened for binding to PT-Gliadin by ELISA. We identified an enriched scFv motif, which showed reactivity to PT-Gliadin. One selected scFv candidate was expressed and purified to homogeneity. Polyclonal anti-PT-Gliadin IgY, purified from egg yolk of immunized chicken, served as control. ScFv binds in a dose-dependent manner to PT-Gliadin, comparable to IgY. Furthermore, IgY competitively displaces scFv from PT-Gliadin and natural wheat flour digest, indicating a common epitope of scFv and IgY. ScFv was tested for reactivity to different gastric digested dietary grain flours. ScFv detects common and khorasan wheat comparably with binding affinities in the high nanomolar range, while rye is detected to a lesser extent. Notably, barley and cereals which are part of the gluten-free diet, like corn and rice, are not detected by scFv. Similarly, the pseudo-grain amaranth, used as gluten-free alternative, is not targeted by scFv. This data indicate that scFv specifically recognizes toxic cereal peptides relevant in CD.

Conclusion: ScFv can be of benefit for future CD treatment regimes.

Show MeSH

Related in: MedlinePlus

Expression time course of scFv which is soluble expressed in E. coli BL21/ (DE3). Crude extracts (30 μg protein per lane) from indicated time points before/after induction with 1 mM IPTG were separated by SDS-PAGE and blotted on PVDF membranes. Western Blot was performed as described in Methods. The upper lane shows target protein expression, the lower lane (E. coli 60 kDa HSP60 protein) represents the loading control. Mock represents the negative control (lysates of bacteria transformed with empty vector). Experiment shown is representative for at least three repeated experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4666168&req=5

Fig4: Expression time course of scFv which is soluble expressed in E. coli BL21/ (DE3). Crude extracts (30 μg protein per lane) from indicated time points before/after induction with 1 mM IPTG were separated by SDS-PAGE and blotted on PVDF membranes. Western Blot was performed as described in Methods. The upper lane shows target protein expression, the lower lane (E. coli 60 kDa HSP60 protein) represents the loading control. Mock represents the negative control (lysates of bacteria transformed with empty vector). Experiment shown is representative for at least three repeated experiments

Mentions: For the production of PT-Gliadin reactive scFvs, we relied on chicken which were immunized three times and showed a respectable antibody blood titer (Fig. 2). Chicken were sacrificed 10–14 days after the third immunization. Splenic lymphocyte fractions were harvested by ficoll gradient centrifugation and mononuclear cells were subjected to magnetic beads previously coated with PT-gliadin. This step was applied to enrich antigen specific B lymphocytes which bind to PT-Gliadin, coated on beads. RNA of beads-bound cells was extracted and cDNA was synthesized. Chicken VH and VL genes were first amplified by PCR with specific primer pairs, followed by amplification in a second PCR step to introduce the linker sequence and sites for restriction digestion. These engineered VH and VL fragments were then ligated into plasmid vectors to generate VH and VL libraries, respectively. VH and VL fragments were excised from the plasmids and ligated into the prokaryotic expression vector pET28a(+), which provides a C-terminal hexahistidine tag (Fig. 3). Screening (at least four clones per plate) revealed that the majority of clones carried correct scFvs (data not shown). Soluble protein expression overtime was assessed by western blot (Fig. 4). ScFvs are soluble expressed in E. coli with increasing target protein concentration over time. The harvesting time point was set to 4–5 h post induction for screening of PT-Gliadin reactive scFvs.Fig. 2


Novel avian single-chain fragment variable (scFv) targets dietary gluten and related natural grain prolamins, toxic entities of celiac disease.

Stadlmann V, Harant H, Korschineck I, Hermann M, Forster F, Missbichler A - BMC Biotechnol. (2015)

Expression time course of scFv which is soluble expressed in E. coli BL21/ (DE3). Crude extracts (30 μg protein per lane) from indicated time points before/after induction with 1 mM IPTG were separated by SDS-PAGE and blotted on PVDF membranes. Western Blot was performed as described in Methods. The upper lane shows target protein expression, the lower lane (E. coli 60 kDa HSP60 protein) represents the loading control. Mock represents the negative control (lysates of bacteria transformed with empty vector). Experiment shown is representative for at least three repeated experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666168&req=5

Fig4: Expression time course of scFv which is soluble expressed in E. coli BL21/ (DE3). Crude extracts (30 μg protein per lane) from indicated time points before/after induction with 1 mM IPTG were separated by SDS-PAGE and blotted on PVDF membranes. Western Blot was performed as described in Methods. The upper lane shows target protein expression, the lower lane (E. coli 60 kDa HSP60 protein) represents the loading control. Mock represents the negative control (lysates of bacteria transformed with empty vector). Experiment shown is representative for at least three repeated experiments
Mentions: For the production of PT-Gliadin reactive scFvs, we relied on chicken which were immunized three times and showed a respectable antibody blood titer (Fig. 2). Chicken were sacrificed 10–14 days after the third immunization. Splenic lymphocyte fractions were harvested by ficoll gradient centrifugation and mononuclear cells were subjected to magnetic beads previously coated with PT-gliadin. This step was applied to enrich antigen specific B lymphocytes which bind to PT-Gliadin, coated on beads. RNA of beads-bound cells was extracted and cDNA was synthesized. Chicken VH and VL genes were first amplified by PCR with specific primer pairs, followed by amplification in a second PCR step to introduce the linker sequence and sites for restriction digestion. These engineered VH and VL fragments were then ligated into plasmid vectors to generate VH and VL libraries, respectively. VH and VL fragments were excised from the plasmids and ligated into the prokaryotic expression vector pET28a(+), which provides a C-terminal hexahistidine tag (Fig. 3). Screening (at least four clones per plate) revealed that the majority of clones carried correct scFvs (data not shown). Soluble protein expression overtime was assessed by western blot (Fig. 4). ScFvs are soluble expressed in E. coli with increasing target protein concentration over time. The harvesting time point was set to 4–5 h post induction for screening of PT-Gliadin reactive scFvs.Fig. 2

Bottom Line: Similarly, the pseudo-grain amaranth, used as gluten-free alternative, is not targeted by scFv.This data indicate that scFv specifically recognizes toxic cereal peptides relevant in CD.ScFv can be of benefit for future CD treatment regimes.

View Article: PubMed Central - PubMed

Affiliation: Sciotec Diagnostics Technologies GmbH, Tulln, Austria. valeriestadlmann@hotmail.com.

ABSTRACT

Background: Celiac disease (CD) is a chronic, small intestinal inflammatory disease mediated by dietary gluten and related prolamins. The only current therapeutic option is maintenance of a strict life-long gluten-free diet, which implies substantial burden for CD patients. Different treatment regimes might be feasible, including masking of toxic celiac peptides with blocking antibodies or fragments thereof. The objective of this study was therefore to select and produce a recombinant avian single-chain fragment variable (scFv) directed against peptic-tryptic digested gliadin (PT-Gliadin) and related celiac toxic entities.

Results: Gluten-free raised chicken of same age were immunized with PT-Gliadin. Chicken splenic lymphocytes, selected with antigen-coated magnetic beads, served as RNA source for the generation of cDNA. Chicken VH and VL genes were amplified from the cDNA by PCR to generate full-length scFv constructs consisting of VH and VL fragments joined by a linker sequence. ScFv constructs were ligated in a prokaryotic expression vector, which provides a C-terminal hexahistidine tag. ScFvs from several bacterial clones were expressed in soluble form and crude cell lysates screened for binding to PT-Gliadin by ELISA. We identified an enriched scFv motif, which showed reactivity to PT-Gliadin. One selected scFv candidate was expressed and purified to homogeneity. Polyclonal anti-PT-Gliadin IgY, purified from egg yolk of immunized chicken, served as control. ScFv binds in a dose-dependent manner to PT-Gliadin, comparable to IgY. Furthermore, IgY competitively displaces scFv from PT-Gliadin and natural wheat flour digest, indicating a common epitope of scFv and IgY. ScFv was tested for reactivity to different gastric digested dietary grain flours. ScFv detects common and khorasan wheat comparably with binding affinities in the high nanomolar range, while rye is detected to a lesser extent. Notably, barley and cereals which are part of the gluten-free diet, like corn and rice, are not detected by scFv. Similarly, the pseudo-grain amaranth, used as gluten-free alternative, is not targeted by scFv. This data indicate that scFv specifically recognizes toxic cereal peptides relevant in CD.

Conclusion: ScFv can be of benefit for future CD treatment regimes.

Show MeSH
Related in: MedlinePlus