Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics

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Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics. / Brofelth, Mattias; Städe, Lars Wagner; Ekstrand, Anna Isinger; Petersson Edfeldt, Linn; Kovačič, Rebeka; Nielsen, Thorbjørn Terndrup; Larsen, Kim Lambertsen; Duroux, Laurent; Wingren, Christer.

In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1865, No. 8, 08.2017, p. 985-996.

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Brofelth, Mattias ; Städe, Lars Wagner ; Ekstrand, Anna Isinger ; Petersson Edfeldt, Linn ; Kovačič, Rebeka ; Nielsen, Thorbjørn Terndrup ; Larsen, Kim Lambertsen ; Duroux, Laurent ; Wingren, Christer. / Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics. In: Biochimica et Biophysica Acta - Proteins and Proteomics. 2017 ; Vol. 1865, No. 8. pp. 985-996.

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TY - JOUR

T1 - Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics

AU - Brofelth, Mattias

AU - Städe, Lars Wagner

AU - Ekstrand, Anna Isinger

AU - Petersson Edfeldt, Linn

AU - Kovačič, Rebeka

AU - Nielsen, Thorbjørn Terndrup

AU - Larsen, Kim Lambertsen

AU - Duroux, Laurent

AU - Wingren, Christer

PY - 2017/8

Y1 - 2017/8

N2 - Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized β-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics.

AB - Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized β-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics.

KW - Antibody engineering

KW - Antibody microarray

KW - Dock'n'Flash

KW - Photochemistry

KW - ScFv antibodies

KW - Site-specific coupling

KW - Unnatural amino acids

UR - http://www.scopus.com/inward/record.url?scp=85019741001&partnerID=8YFLogxK

U2 - 10.1016/j.bbapap.2017.03.007

DO - 10.1016/j.bbapap.2017.03.007

M3 - Article

VL - 1865

SP - 985

EP - 996

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 8

ER -