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

Brofelth, Mattias LU ; Städe, Lars Wagner; Ekstrand, Anna Isinger LU ; Petersson Edfeldt, Linn LU ; Kovačič, Rebeka; Nielsen, Thorbjørn Terndrup; Larsen, Kim Lambertsen; Duroux, Laurent and Wingren, Christer LU (2017) In Biochimica et Biophysica Acta - Proteins and Proteomics 1865(8). p.985-996
Abstract

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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Antibody engineering, Antibody microarray, Dock'n'Flash, Photochemistry, ScFv antibodies, Site-specific coupling, Unnatural amino acids
in
Biochimica et Biophysica Acta - Proteins and Proteomics
volume
1865
issue
8
pages
12 pages
publisher
Elsevier
external identifiers
  • scopus:85019741001
  • wos:000404323400002
ISSN
1570-9639
DOI
10.1016/j.bbapap.2017.03.007
language
English
LU publication?
yes
id
4cb01d5c-dd8a-4568-94d4-b3606cbabb54
date added to LUP
2017-06-09 14:48:37
date last changed
2017-09-18 11:40:05
@article{4cb01d5c-dd8a-4568-94d4-b3606cbabb54,
  abstract     = {<p>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.</p>},
  author       = {Brofelth, Mattias and Städe, Lars Wagner and Ekstrand, Anna Isinger and Petersson Edfeldt, Linn and Kovačič, Rebeka and Nielsen, Thorbjørn Terndrup and Larsen, Kim Lambertsen and Duroux, Laurent and Wingren, Christer},
  issn         = {1570-9639},
  keyword      = {Antibody engineering,Antibody microarray,Dock'n'Flash,Photochemistry,ScFv antibodies,Site-specific coupling,Unnatural amino acids},
  language     = {eng},
  number       = {8},
  pages        = {985--996},
  publisher    = {Elsevier},
  series       = {Biochimica et Biophysica Acta - Proteins and Proteomics},
  title        = {Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics},
  url          = {http://dx.doi.org/10.1016/j.bbapap.2017.03.007},
  volume       = {1865},
  year         = {2017},
}