Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment.
Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are ready targets for labelling by an isothiocyanate derivative of the europium chelate (Eu3+). Here the labelling efficiency of a recombinant anti-human alpha-fetoprotein (hAFP) Fab fragment has been improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues, R154, R187 and R210, which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment and its labelling efficiency was found to be approximately 40% higher than that of the wild-type Fab fragment. With low degree of labelling, the affinities of the two Fab fragments were identical and comparable with that of the original monoclonal anti-hAFP IgG. With a higher degree of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. Electrostatic adsorption and covalent immobilization of the Fab fragments were characterized by BIAcore and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.
Hemminki
A
A
VTT Biotechnology and Food Research, Espoo, Finland.
Hoffrén
A M
AM
Takkinen
K
K
Vehniäinen
M
M
Mäkinen
M L
ML
Pettersson
K
K
Teleman
O
O
Söderlund
H
H
Teeri
T T
TT
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001483:Base Sequence; D003001:Cloning, Molecular; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics; D005063:Europium; D005456:Fluorescent Dyes; D015200:Fluoroimmunoassay; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008239:Lysine; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis; D000509:alpha-Fetoproteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; D005063:Europium; D008239:Lysine