INTRODUCTION OF LYSINE RESIDUES ON THE LIGHT-CHAIN CONSTANT DOMAIN IMPROVES THE LABELING PROPERTIES OF A RECOMBINANT FAB FRAGMENT

A HEMMINKI*, AM HOFFREN, K TAKKINEN, M VEHNIAINEN, ML MAKINEN, K PETTERSSON, O TELEMAN, H SODERLUND, Tuula T. Teeri

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

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 (EU(3+)). Here the labelling efficiency of a recombinant anti-human a-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 similar to 40% higher than that of the wildtype 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(TM) and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.

Original languageEnglish
Pages (from-to)185-191
Number of pages7
JournalPROTEIN ENGINEERING
Volume8
Issue number2
Publication statusPublished - Feb 1995
MoE publication typeA1 Journal article-refereed

Keywords

  • EU(3+) LABELING
  • MOLECULAR MODELING
  • RECOMBINANT FAB FRAGMENT
  • SITE-DIRECTED MUTAGENESIS
  • SURFACE RESIDUES
  • BIOSPECIFIC INTERACTION ANALYSIS
  • SURFACE-PLASMON RESONANCE
  • ESCHERICHIA-COLI
  • FV FRAGMENT
  • MONOCLONAL-ANTIBODIES
  • HOMOLOGOUS PROTEINS
  • DATA-BANK
  • EXPRESSION
  • POLYMERASE
  • SECRETION

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