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


CONCLUSIONS
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.
Reulen
Sanne W A
SW
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
van Baal
Ingrid
I
Raats
Jos M H
JM
Merkx
Maarten
M
D000906:Antibodies / Q000737:chemistry* / Q000302:isolation & purification; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D047668:Inteins; D015080:Mesna / Q000737:chemistry; D008823:Micelles; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification
D000906:Antibodies; D008823:Micelles; D011993:Recombinant Fusion Proteins; D003545:Cysteine; D015080:Mesna