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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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The binding of the sdAb-aGST-MESNA to GST assayed with SPR and ELISA. (A) SPR analysis of sdAb-aGST-MESNA binding to GST. Insert: steady state responses were plotted against the concentration. The line represents a fit to a one-site binding model using a KD of 116 nM. (B) ELISA measurement of binding of sdAb-aGST to GST (square) fitted with a one-site binding model using a KD of 77 nM and BSA (circle).
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Figure 3: The binding of the sdAb-aGST-MESNA to GST assayed with SPR and ELISA. (A) SPR analysis of sdAb-aGST-MESNA binding to GST. Insert: steady state responses were plotted against the concentration. The line represents a fit to a one-site binding model using a KD of 116 nM. (B) ELISA measurement of binding of sdAb-aGST to GST (square) fitted with a one-site binding model using a KD of 77 nM and BSA (circle).

Mentions: The sdAb-aGST protein contains a single disulfide bond that has previously been shown to play an essential role in the stability of these single-domain antibodies. To establish whether the reducing conditions used during MESNA cleavage affected the sdAb-aGST-MESNA produced in this manner, its interaction with GST was studied using Surface Plasmon Resonance (SPR). Initial attempts to immobilize the sdAb-aGST-MESNA to the CM5 Biacore chip using a standard amine coupling procedure resulted in a complete loss of GST binding capacity, suggesting that this classical amine coupling affects residues critical for antigen binding. The sdAb-aGST-MESNA protein contains 11 lysine residues, two of which are indeed located near the antigen binding site. To exclude antibody deactivation the assay was reversed and GST was immobilized on the CM5 chip. Figure 3A shows a clear binding response of the sdAb-aGST-MESNA to GST with an apparent dissociation constant of 120 nM. This number is in good agreement with results from an ELISA assay, which showed a dissociation constant of 80 nM (Figure 3B). The observed dissociation constants for the sdAb-aGST are similar to those reported for other single-domain antibodies [32]. In addition, SPR analysis of sdAb-aGST-MESNA after NCL with cysteine (sdAb-aGST-Cys) showed binding behavior that was similar to that of untreated sdAb-aGST (see Additional file 1). So while we cannot exclude at this time that the disulfide bond becomes transiently reduced during MESNA treatment or NCL, these procedures did not irreversibly affect the functionality of sdAb-GST.


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)

The binding of the sdAb-aGST-MESNA to GST assayed with SPR and ELISA. (A) SPR analysis of sdAb-aGST-MESNA binding to GST. Insert: steady state responses were plotted against the concentration. The line represents a fit to a one-site binding model using a KD of 116 nM. (B) ELISA measurement of binding of sdAb-aGST to GST (square) fitted with a one-site binding model using a KD of 77 nM and BSA (circle).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719619&req=5

Figure 3: The binding of the sdAb-aGST-MESNA to GST assayed with SPR and ELISA. (A) SPR analysis of sdAb-aGST-MESNA binding to GST. Insert: steady state responses were plotted against the concentration. The line represents a fit to a one-site binding model using a KD of 116 nM. (B) ELISA measurement of binding of sdAb-aGST to GST (square) fitted with a one-site binding model using a KD of 77 nM and BSA (circle).
Mentions: The sdAb-aGST protein contains a single disulfide bond that has previously been shown to play an essential role in the stability of these single-domain antibodies. To establish whether the reducing conditions used during MESNA cleavage affected the sdAb-aGST-MESNA produced in this manner, its interaction with GST was studied using Surface Plasmon Resonance (SPR). Initial attempts to immobilize the sdAb-aGST-MESNA to the CM5 Biacore chip using a standard amine coupling procedure resulted in a complete loss of GST binding capacity, suggesting that this classical amine coupling affects residues critical for antigen binding. The sdAb-aGST-MESNA protein contains 11 lysine residues, two of which are indeed located near the antigen binding site. To exclude antibody deactivation the assay was reversed and GST was immobilized on the CM5 chip. Figure 3A shows a clear binding response of the sdAb-aGST-MESNA to GST with an apparent dissociation constant of 120 nM. This number is in good agreement with results from an ELISA assay, which showed a dissociation constant of 80 nM (Figure 3B). The observed dissociation constants for the sdAb-aGST are similar to those reported for other single-domain antibodies [32]. In addition, SPR analysis of sdAb-aGST-MESNA after NCL with cysteine (sdAb-aGST-Cys) showed binding behavior that was similar to that of untreated sdAb-aGST (see Additional file 1). So while we cannot exclude at this time that the disulfide bond becomes transiently reduced during MESNA treatment or NCL, these procedures did not irreversibly affect the functionality of sdAb-GST.

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