Molecular design of recombinant scFv antibodies for site-specific photocoupling to β-cyclodextrin in solution and onto solid support.

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Molecular design of recombinant scFv antibodies for site-specific photocoupling to β-cyclodextrin in solution and onto solid support. / Petersson, Linn; Städe, Lars Wagner; Brofelth, Mattias; Gärtner, Stefanie; Fors, Elin; Sandgren, Martin; Vallkil, Jacob; Olsson, Niclas; Larsen, Kim Lambertsen; Borrebaeck, Carl; Duroux, Laurent; Wingren, Christer.

I: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1844, Nr. 12, 2014, s. 2164-2173.

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

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Petersson, Linn ; Städe, Lars Wagner ; Brofelth, Mattias ; Gärtner, Stefanie ; Fors, Elin ; Sandgren, Martin ; Vallkil, Jacob ; Olsson, Niclas ; Larsen, Kim Lambertsen ; Borrebaeck, Carl ; Duroux, Laurent ; Wingren, Christer. / Molecular design of recombinant scFv antibodies for site-specific photocoupling to β-cyclodextrin in solution and onto solid support. I: Biochimica et Biophysica Acta - Proteins and Proteomics. 2014 ; Vol. 1844, Nr. 12. s. 2164-2173.

RIS

TY - JOUR

T1 - Molecular design of recombinant scFv antibodies for site-specific photocoupling to β-cyclodextrin in solution and onto solid support.

AU - Petersson, Linn

AU - Städe, Lars Wagner

AU - Brofelth, Mattias

AU - Gärtner, Stefanie

AU - Fors, Elin

AU - Sandgren, Martin

AU - Vallkil, Jacob

AU - Olsson, Niclas

AU - Larsen, Kim Lambertsen

AU - Borrebaeck, Carl

AU - Duroux, Laurent

AU - Wingren, Christer

PY - 2014

Y1 - 2014

N2 - The ability to design and tailor-make antibodies to meet the biophysical demands required by the vast range of current and future antibody-based applications within biotechnology and biomedicine will be essential. In this proof-of-concept study, we have for the first time tailored 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 successfully explored the possibility to expand the genetic code of E. coli and introduced the photoreactive UAA p-benzoyl-L-phenylalanine (pBpa), and showed that the mutated scFv antibody could be expressed in E. coli with retained structural and functional properties, as well as binding affinity. The pBpa group was then used for affinity capture of the mutated antibody by β-cyclodextrin (β-CD), which provided the hydrogen atoms to be abstracted in the subsequent photocoupling process upon irradiation at 365nm. The results showed that the pBpa mutated antibody could be site-specifically photocoupled to free and surface (array) immobilized β-CD. Taken together, this paves the way for novel means of tailoring recombinant scFv antibodies for site-specific photochemical-based tagging, functionalization and immobilization in numerous applications.

AB - The ability to design and tailor-make antibodies to meet the biophysical demands required by the vast range of current and future antibody-based applications within biotechnology and biomedicine will be essential. In this proof-of-concept study, we have for the first time tailored 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 successfully explored the possibility to expand the genetic code of E. coli and introduced the photoreactive UAA p-benzoyl-L-phenylalanine (pBpa), and showed that the mutated scFv antibody could be expressed in E. coli with retained structural and functional properties, as well as binding affinity. The pBpa group was then used for affinity capture of the mutated antibody by β-cyclodextrin (β-CD), which provided the hydrogen atoms to be abstracted in the subsequent photocoupling process upon irradiation at 365nm. The results showed that the pBpa mutated antibody could be site-specifically photocoupled to free and surface (array) immobilized β-CD. Taken together, this paves the way for novel means of tailoring recombinant scFv antibodies for site-specific photochemical-based tagging, functionalization and immobilization in numerous applications.

U2 - 10.1016/j.bbapap.2014.08.010

DO - 10.1016/j.bbapap.2014.08.010

M3 - Article

VL - 1844

SP - 2164

EP - 2173

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

T2 - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 12

ER -