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

Ari Hemminki (Corresponding Author), Anna-Marja Hoffren, Kristiina Takkinen, Markus Vehniäinen, Maija-Liisa Mäkinen, Kim Pettersson, Olle Teleman, Hans Söderlund, Tuula Teeri

Research output: Contribution to journalArticleScientificpeer-review

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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 (Eu3+). Here the labelling efficiency of a recombinant anti-human α-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 ∼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 BIAcoreTM 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
JournalProtein Engineering
Volume8
Issue number2
DOIs
Publication statusPublished - 1995
MoE publication typeA1 Journal article-refereed

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Immunoglobulin Fab Fragments
Labeling
Lysine
Light
Fetal Proteins
Europium
Antibodies
Immunoglobulin G
Protein Engineering
Arginine
Molecular modeling
Static Electricity
Immunoassay
Immobilization
Adsorption
Electrostatics
Derivatives
Proteins
Mutation
Molecules

Cite this

Hemminki, Ari ; Hoffren, Anna-Marja ; Takkinen, Kristiina ; Vehniäinen, Markus ; Mäkinen, Maija-Liisa ; Pettersson, Kim ; Teleman, Olle ; Söderlund, Hans ; Teeri, Tuula. / Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment. In: Protein Engineering. 1995 ; Vol. 8, No. 2. pp. 185-191.
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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 (Eu3+). Here the labelling efficiency of a recombinant anti-human α-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 ∼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 BIAcoreTM and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.",
author = "Ari Hemminki and Anna-Marja Hoffren and Kristiina Takkinen and Markus Vehni{\"a}inen and Maija-Liisa M{\"a}kinen and Kim Pettersson and Olle Teleman and Hans S{\"o}derlund and Tuula Teeri",
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Hemminki, A, Hoffren, A-M, Takkinen, K, Vehniäinen, M, Mäkinen, M-L, Pettersson, K, Teleman, O, Söderlund, H & Teeri, T 1995, 'Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment', Protein Engineering, vol. 8, no. 2, pp. 185-191. https://doi.org/10.1093/protein/8.2.185

Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment. / Hemminki, Ari (Corresponding Author); Hoffren, Anna-Marja; Takkinen, Kristiina; Vehniäinen, Markus; Mäkinen, Maija-Liisa; Pettersson, Kim; Teleman, Olle; Söderlund, Hans; Teeri, Tuula.

In: Protein Engineering, Vol. 8, No. 2, 1995, p. 185-191.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment

AU - Hemminki, Ari

AU - Hoffren, Anna-Marja

AU - Takkinen, Kristiina

AU - Vehniäinen, Markus

AU - Mäkinen, Maija-Liisa

AU - Pettersson, Kim

AU - Teleman, Olle

AU - Söderlund, Hans

AU - Teeri, Tuula

N1 - Project code: BEL2019

PY - 1995

Y1 - 1995

N2 - 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 α-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 ∼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 BIAcoreTM and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.

AB - 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 α-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 ∼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 BIAcoreTM and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.

U2 - 10.1093/protein/8.2.185

DO - 10.1093/protein/8.2.185

M3 - Article

VL - 8

SP - 185

EP - 191

JO - Protein Engineering, Design and Selection

JF - Protein Engineering, Design and Selection

SN - 1741-0126

IS - 2

ER -