Year-end Sale % OFF 
Abstract
BackgroundClassical 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.ResultsTargeting 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.ConclusionA 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.
Highlights
Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity
Production of single-domain antibodies with a C-terminal thioester Our approach, schematically depicted in Figure 1, was tested using a llama single-domain antibody obtained from screening a phage display library against glutathione-S from Schistosoma japonicum
The pelB leader sequence was used to target the protein to the oxidizing environment of the periplasm, because the single-domain antibodies (sdAb)-aGST protein contains a conserved disulfide bond that is known to be important for the stability of these single domain antibodies [29]
Summary
Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. The ability to raise antibodies with high affinity and specificity to almost any biomolecular target has made antibodies essential components in many biomedical fields, both in diagnostics and in the active targeting of drugs and contrast agents for molecular imaging [1]. For many of these applications there has been a drive to move towards smaller antibody formats, both to allow efficient recombinant production in E. coli and to potentially avoid unwanted immunogenic problems [2]. Single-domain antibodies are the smallest antibody fragments available to date and have unique features including high solubility and thermal stability [4]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
