title: Multiple site-specific in vitro labeling of single-chain antibody.
abstract: For multiple site-specific conjugations of bioactive molecules to a single-chain antibody (scFv) molecule, we have constructed a human anti HER2 receptor, scFv, with a C-terminal fusion polypeptide containing 1, 3, or 17 threonine (Thr) residues. The C-terminal extended fusion polypeptides of these recombinant scFv fusion proteins are used as the acceptor substrate for human polypeptide-alpha-Nu-acetylgalactosaminyltransferase II (h-ppGalNAc-T2) that transfers either GalNAc or 2-keto-Gal, a modified galactose with a chemical handle, from their respective UDP-sugars to the side-chain hydroxyl group of the Thr residue(s). The recombinant scFv fusion proteins are expressed in E. coli as inclusion bodies and in vitro refolded and glycosylated with h-ppGalNAc-T2. Upon protease cleavage, the MALDI-TOF spectra of the glycosylated C-terminal fusion polypeptides showed that the glycosylated scFv fusion protein with a single Thr residue is fully glycosylated with a single 2-keto-Gal, whereas the glycosylated scFv fusion protein with 3 and 17 Thr residues is found as an equal mixture of 2-3 and 5-8 2-keto-Gal glycosylated fusion proteins, respectively. These fusion scFv proteins with the modified galactose are then conjugated with a fluorescence probe, Alexa488, that carries an orthogonal reactive group. The fluorescence labeled scFv proteins bind specifically to a human breast cancer cell line (SK-BR-3) that overexpresses the HER2 receptor, indicating that the in vitro folded scFv fusion proteins are biologically active and the presence of conjugated multiple Alexa488 probes in their C-terminal end does not interfere with their binding to the antigen.
authors.affiliation: Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, Maryland 21702, USA.
authors.forename: Boopathy
authors.initials: B
authors.lastname: Ramakrishnan
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Boeggeman
authors.forename: Maria
authors.initials: M
authors.lastname: Manzoni
authors.forename: Zhongyu
authors.initials: Z
authors.lastname: Zhu
authors.forename: Kristin
authors.initials: K
authors.lastname: Loomis
authors.forename: Anu
authors.initials: A
authors.lastname: Puri
authors.forename: Dimiter S
authors.initials: DS
authors.lastname: Dimitrov
authors.forename: Pradman K
authors.initials: PK
authors.lastname: Qasba
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human