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  • Tailor-making a protein a-derived domain for efficient site-specific photocoupling to Fc of mouse IgG₁.

Tailor-making a protein a-derived domain for efficient site-specific photocoupling to Fc of mouse IgG₁.

PloS one (2013-02-21)
Feifan Yu, Peter Järver, Per-Åke Nygren
ABSTRACT

Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG₁ (mIgG₁). Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG₁ monoclonal antibodies (mAbs). The best variant, denoted Z(F5I) corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z(F5I) variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP) group as a probe for site-specific photoconjugation to Fc of mIgG₁, The best photocoupling efficiency to mIgG₁ Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG₁ mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG₁ antibody via site-specific biotinylation, the Z(F5I-Q32C-MBP) protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG₁ mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly higher bioactivity was observed for the sample biotinylated using the Z(F5I-Q32C-MBP) probe. This result indicates that the use of a site-specific and affinity probe-mediated conjugation strategy can result in antibody reagents with increased assay sensitivity.

MATERIALS
Product Number
Brand
Product Description

Supelco
Mettler-Toledo Calibration substance ME 18870, Benzophenone, traceable to primary standards (LGC)
Supelco
Melting point standard 47-49°C, analytical standard
Sigma-Aldrich
Benzophenone, purified by sublimation, ≥99%
Supelco
Benzophenone, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Benzophenone, analytical standard
Sigma-Aldrich
Benzophenone, ReagentPlus®, 99%
Benzophenone, European Pharmacopoeia (EP) Reference Standard