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Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester.

Reulen SW, van Baal I, Raats JM, Merkx M - BMC Biotechnol. (2009)

Bottom Line: Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity.However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, the Netherlands. s.w.a.reulen@tue.nl

ABSTRACT

Background: Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.

Results: Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb)-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA)-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step.

Conclusion: A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

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Chromatogram and deconvoluted mass spectrum of the biotinylated single-domain antibody against GST after overnight ligation. pelB-biotin: biotinylated sdAb-aGST with pelB leader attached (calcd. mass 19879 Da); pelB: MESNA thioester of the sdAb-aGST with pelB leader attached (calcd. mass 19632 Da); pelBΔ3-biotin: biotinylated sdAb-aGST with the first three amino acids of the pelB leader removed (calcd. mass 19457 Da);); pelBΔ14-biotin: biotinylated sdAb-aGST with the first 14 amino acids of the pelB leader removed (calcd. mass 18423 Da);pelBΔ15-biotin: biotinylated sdAb-aGST with the first 15 amino acids of the pelB leader removed (calcd. mass 18309 Da); pelBΔ16-biotin: biotinylated sdAb-aGST with the first 16 amino acids of the pelB leader removed (calcd. mass 18238 Da);
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Figure 4: Chromatogram and deconvoluted mass spectrum of the biotinylated single-domain antibody against GST after overnight ligation. pelB-biotin: biotinylated sdAb-aGST with pelB leader attached (calcd. mass 19879 Da); pelB: MESNA thioester of the sdAb-aGST with pelB leader attached (calcd. mass 19632 Da); pelBΔ3-biotin: biotinylated sdAb-aGST with the first three amino acids of the pelB leader removed (calcd. mass 19457 Da);); pelBΔ14-biotin: biotinylated sdAb-aGST with the first 14 amino acids of the pelB leader removed (calcd. mass 18423 Da);pelBΔ15-biotin: biotinylated sdAb-aGST with the first 15 amino acids of the pelB leader removed (calcd. mass 18309 Da); pelBΔ16-biotin: biotinylated sdAb-aGST with the first 16 amino acids of the pelB leader removed (calcd. mass 18238 Da);

Mentions: To test the performance of sdAb-aGST-MESNA in NCL reactions, we first assessed its reactivity towards cysteine-functionalized biotin. Ligation of this small molecule can be conveniently monitored using LC-MS. In addition, single-domain antibodies with C-terminal biotin groups are attractive for use in combination with the many streptavidin-modified compounds that are now commercially available. Figure 4 shows the LC-MS analysis of the product obtained after reaction of sdAb-aGST-MESNA with an excess of cysteine-functionalized biotin overnight at room temperature. Again two peaks appear in the chromatogram corresponding to sdAb-aGST with various fragments of the pelB leader sequence still attached. All peaks correspond to biotinylated forms of sdAb-aGST, with the exception of a minor peak at 19635 Da corresponding to a small amount of unreacted sdAb-aGST-MESNA.


Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester.

Reulen SW, van Baal I, Raats JM, Merkx M - BMC Biotechnol. (2009)

Chromatogram and deconvoluted mass spectrum of the biotinylated single-domain antibody against GST after overnight ligation. pelB-biotin: biotinylated sdAb-aGST with pelB leader attached (calcd. mass 19879 Da); pelB: MESNA thioester of the sdAb-aGST with pelB leader attached (calcd. mass 19632 Da); pelBΔ3-biotin: biotinylated sdAb-aGST with the first three amino acids of the pelB leader removed (calcd. mass 19457 Da);); pelBΔ14-biotin: biotinylated sdAb-aGST with the first 14 amino acids of the pelB leader removed (calcd. mass 18423 Da);pelBΔ15-biotin: biotinylated sdAb-aGST with the first 15 amino acids of the pelB leader removed (calcd. mass 18309 Da); pelBΔ16-biotin: biotinylated sdAb-aGST with the first 16 amino acids of the pelB leader removed (calcd. mass 18238 Da);
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2719619&req=5

Figure 4: Chromatogram and deconvoluted mass spectrum of the biotinylated single-domain antibody against GST after overnight ligation. pelB-biotin: biotinylated sdAb-aGST with pelB leader attached (calcd. mass 19879 Da); pelB: MESNA thioester of the sdAb-aGST with pelB leader attached (calcd. mass 19632 Da); pelBΔ3-biotin: biotinylated sdAb-aGST with the first three amino acids of the pelB leader removed (calcd. mass 19457 Da);); pelBΔ14-biotin: biotinylated sdAb-aGST with the first 14 amino acids of the pelB leader removed (calcd. mass 18423 Da);pelBΔ15-biotin: biotinylated sdAb-aGST with the first 15 amino acids of the pelB leader removed (calcd. mass 18309 Da); pelBΔ16-biotin: biotinylated sdAb-aGST with the first 16 amino acids of the pelB leader removed (calcd. mass 18238 Da);
Mentions: To test the performance of sdAb-aGST-MESNA in NCL reactions, we first assessed its reactivity towards cysteine-functionalized biotin. Ligation of this small molecule can be conveniently monitored using LC-MS. In addition, single-domain antibodies with C-terminal biotin groups are attractive for use in combination with the many streptavidin-modified compounds that are now commercially available. Figure 4 shows the LC-MS analysis of the product obtained after reaction of sdAb-aGST-MESNA with an excess of cysteine-functionalized biotin overnight at room temperature. Again two peaks appear in the chromatogram corresponding to sdAb-aGST with various fragments of the pelB leader sequence still attached. All peaks correspond to biotinylated forms of sdAb-aGST, with the exception of a minor peak at 19635 Da corresponding to a small amount of unreacted sdAb-aGST-MESNA.

Bottom Line: Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity.However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, the Netherlands. s.w.a.reulen@tue.nl

ABSTRACT

Background: Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.

Results: Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb)-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA)-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step.

Conclusion: A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

Show MeSH