Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment. 1995

A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
VTT Biotechnology and Food Research, Espoo, Finland.

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.

UI MeSH Term Description Entries
D007140 Immunoglobulin Fab Fragments Univalent antigen-binding fragments composed of one entire IMMUNOGLOBULIN LIGHT CHAIN and the amino terminal end of one of the IMMUNOGLOBULIN HEAVY CHAINS from the hinge region, linked to each other by disulfide bonds. Fab contains the IMMUNOGLOBULIN VARIABLE REGIONS, which are part of the antigen-binding site, and the first IMMUNOGLOBULIN CONSTANT REGIONS. This fragment can be obtained by digestion of immunoglobulins with the proteolytic enzyme PAPAIN. Fab Fragment,Fab Fragments,Ig Fab Fragments,Immunoglobulins, Fab Fragment,Fab Immunoglobulin Fragments,Immunoglobulin Fab Fragment,Immunoglobulins, Fab,Fab Fragment Immunoglobulins,Fab Fragment, Immunoglobulin,Fab Fragments, Immunoglobulin,Fragment Immunoglobulins, Fab,Fragment, Fab,Immunoglobulin Fragments, Fab
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008239 Lysine An essential amino acid. It is often added to animal feed. Enisyl,L-Lysine,Lysine Acetate,Lysine Hydrochloride,Acetate, Lysine,L Lysine
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004563 Electrochemistry The study of chemical changes resulting from electrical action and electrical activity resulting from chemical changes. Electrochemistries
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005063 Europium An element of the rare earth family of metals. It has the atomic symbol Eu, atomic number 63, and atomic weight 152. Europium is used in the form of its salts as coatings for cathode ray tubes and in the form of its organic derivatives as shift reagents in NMR spectroscopy.

Related Publications

A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
September 1987, Journal of biochemistry,
A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
January 2020, Biochimica et biophysica acta. Proteins and proteomics,
A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
April 1987, Biochemistry,
A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
June 2021, Scientific reports,
A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
April 1982, Journal of molecular biology,
A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
August 2010, FEBS letters,
A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
June 2008, Acta crystallographica. Section D, Biological crystallography,
A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
November 1986, Proceedings of the National Academy of Sciences of the United States of America,
A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
May 2010, Biotechnology and bioengineering,
A Hemminki, and A M Hoffrén, and K Takkinen, and M Vehniäinen, and M L Mäkinen, and K Pettersson, and O Teleman, and H Söderlund, and T T Teeri
August 1998, Proceedings of the National Academy of Sciences of the United States of America,
Copied contents to your clipboard!