High yield, site-specific coupling of N-terminally modified beta-lactamase to a proteolytically derived single-sulfhydryl murine Fab'.
Bioconjug Chem
; 5(6): 636-46, 1994.
Article
em En
| MEDLINE
| ID: mdl-7873667
ABSTRACT
The preparation of bispecific protein conjugates capable of performing diverse biological functions is an area of active investigation. Such conjugates are routinely prepared using techniques which employ random derivatization of lysine residues, but the overall utility of these methods is limited due to poor yields and heterogeneous conjugates. In this report we describe the development of site-specific linkage methodology for the chemical synthesis of a homogeneous enzyme-antibody Fab' conjugate with coupling efficiencies of at least 72%. The N-terminal threonine residue of beta-lactamase from the P99 strain of Enterobacter cloacae was oxidized to an aldehyde functional group under mild conditions with a 5-fold molar excess of sodium periodate. The murine Fab' with a single sulfhydryl at the hinge region was generated by further digestion of the peptic Fab' fragment with lysyl endopeptidase to remove a decapeptide containing two of the three cysteine residues. Coupling of the two modified proteins was accomplished through a bifunctional coupling reagent containing meleimide and aminooxy functional groups. Synthesis of the linker is described. Yields of 11 enzyme-Fab' were at least three times higher than for comparable random derivatization methods. Immunoreactivity and enzymatic activity were unaffected. Biodistribution studies showed a more favorable tumor to blood ratio with the site-specifically linked conjugate.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Beta-Lactamases
/
Fragmentos Fab das Imunoglobulinas
Limite:
Animals
Idioma:
En
Revista:
Bioconjug Chem
Assunto da revista:
BIOQUIMICA
Ano de publicação:
1994
Tipo de documento:
Article