Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions

Eeva-Christine Brockmann (Corresponding Author), Matthew Cooper, Nelli Strömsten, Markus Vehniäinen, Petri Saviranta

Research output: Contribution to journalArticleScientificpeer-review

44 Citations (Scopus)

Abstract

Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8–9 °C improved thermal stability and 0.8–0.9 M improved stability for guanidinium chloride were found after 4–5 enrichment cycles. The most stable mutants selected contained either LysH66Arg or AsnH52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.
Original languageEnglish
Pages (from-to)159 - 170
Number of pages12
JournalJournal of Immunological Methods
Volume296
Issue number1-2
DOIs
Publication statusPublished - 2005
MoE publication typeA1 Journal article-refereed

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Immunoglobulin Fragments
Bacteriophages
Guanidine
Thyrotropin
Mutagenesis
Disulfides
Clone Cells
Hot Temperature
Mutation

Keywords

  • Antibody engineering
  • Disulfide bond
  • Phage display
  • Protein denaturation
  • Protein stability

Cite this

Brockmann, Eeva-Christine ; Cooper, Matthew ; Strömsten, Nelli ; Vehniäinen, Markus ; Saviranta, Petri. / Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions. In: Journal of Immunological Methods. 2005 ; Vol. 296, No. 1-2. pp. 159 - 170.
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abstract = "Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8–9 °C improved thermal stability and 0.8–0.9 M improved stability for guanidinium chloride were found after 4–5 enrichment cycles. The most stable mutants selected contained either LysH66Arg or AsnH52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.",
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Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions. / Brockmann, Eeva-Christine (Corresponding Author); Cooper, Matthew; Strömsten, Nelli; Vehniäinen, Markus; Saviranta, Petri.

In: Journal of Immunological Methods, Vol. 296, No. 1-2, 2005, p. 159 - 170.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions

AU - Brockmann, Eeva-Christine

AU - Cooper, Matthew

AU - Strömsten, Nelli

AU - Vehniäinen, Markus

AU - Saviranta, Petri

PY - 2005

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N2 - Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8–9 °C improved thermal stability and 0.8–0.9 M improved stability for guanidinium chloride were found after 4–5 enrichment cycles. The most stable mutants selected contained either LysH66Arg or AsnH52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.

AB - Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8–9 °C improved thermal stability and 0.8–0.9 M improved stability for guanidinium chloride were found after 4–5 enrichment cycles. The most stable mutants selected contained either LysH66Arg or AsnH52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.

KW - Antibody engineering

KW - Disulfide bond

KW - Phage display

KW - Protein denaturation

KW - Protein stability

U2 - 10.1016/j.jim.2004.11.008

DO - 10.1016/j.jim.2004.11.008

M3 - Article

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JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

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