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.
(Less)
- author
- 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
- organization
- publishing date
- 2017-08
- 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
-
- pmid:28342851
- wos:000404323400002
- scopus:85019741001
- 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
- 2025-01-07 15:02:26
@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}},
keywords = {{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}},
doi = {{10.1016/j.bbapap.2017.03.007}},
volume = {{1865}},
year = {{2017}},
}