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Direct Site-Directed Photocoupling of Proteins onto Surfaces Coated with beta-Cyclodextrins

Jensen, Rasmus L.; Stade, Lars W.; Wimmer, Reinhard; Stensballe, Allan; Duroux, Meg; Larsen, Kim L.; Wingren, Christer LU and Duroux, Laurent (2010) In Langmuir 26(13). p.11597-11604
Abstract
A method called Dock'n'Flash was developed to offer site-specific capture and direct UVA-induced photocoupling of recombinant proteins. The method involves the tagging of recombinant proteins with photoreactive p-benzoyl-L-phenylalanine (pBpa) by genetic engineering. The photoreactive pBpa tag is used for affinity capture of the recombinant protein by beta-cyclodextrin (beta-CD), which provides hydrogen atoms to be abstracted in the photocoupling process. To exemplify the method, a recombinant, folded, and active N27pBpa mutant of cutinase from Fusarium solani pisi was produced in E. coli. Insertion of pBpa was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. A inolecular dynamic... (More)
A method called Dock'n'Flash was developed to offer site-specific capture and direct UVA-induced photocoupling of recombinant proteins. The method involves the tagging of recombinant proteins with photoreactive p-benzoyl-L-phenylalanine (pBpa) by genetic engineering. The photoreactive pBpa tag is used for affinity capture of the recombinant protein by beta-cyclodextrin (beta-CD), which provides hydrogen atoms to be abstracted in the photocoupling process. To exemplify the method, a recombinant, folded, and active N27pBpa mutant of cutinase from Fusarium solani pisi was produced in E. coli. Insertion of pBpa was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. A inolecular dynamic simulation, with water as solvent, showed high solvent accessibility of the pBpa benzophenone group in N27pBpa-cutinase mutant. The formation of an inclusion complex between the benzophenone group of N27pBpa-cutinase and beta-CD was shown, and an apparent K-d of 1.65 mM was determined using H-1 NMR. Photocoupling of beta-CD to N27pBpa-cutinase in a 1:1 ratio, upon UVA irradiation at 360 +/- 20 nm, was shown by MALDI-TOF mass spectroscopy. UVA photoimmobilization of N27pBpa-cutinase on quartz slides coated with beta-CD was achieved from liquid or dry films by total internal reflection fluorescence (TIRF). The Dock'n'Flash method offers a solution for direct photocoupling and patterning of recombinant proteins onto surfaces with site-specific attachment, (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Langmuir
volume
26
issue
13
pages
11597 - 11604
publisher
The American Chemical Society
external identifiers
  • wos:000279239900163
  • scopus:77954274027
ISSN
0743-7463
DOI
10.1021/la100950n
language
English
LU publication?
yes
id
a580a9f5-1c5e-4e36-9842-6139ddfe1773 (old id 1629022)
date added to LUP
2010-07-22 11:44:13
date last changed
2018-05-29 11:24:20
@article{a580a9f5-1c5e-4e36-9842-6139ddfe1773,
  abstract     = {A method called Dock'n'Flash was developed to offer site-specific capture and direct UVA-induced photocoupling of recombinant proteins. The method involves the tagging of recombinant proteins with photoreactive p-benzoyl-L-phenylalanine (pBpa) by genetic engineering. The photoreactive pBpa tag is used for affinity capture of the recombinant protein by beta-cyclodextrin (beta-CD), which provides hydrogen atoms to be abstracted in the photocoupling process. To exemplify the method, a recombinant, folded, and active N27pBpa mutant of cutinase from Fusarium solani pisi was produced in E. coli. Insertion of pBpa was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. A inolecular dynamic simulation, with water as solvent, showed high solvent accessibility of the pBpa benzophenone group in N27pBpa-cutinase mutant. The formation of an inclusion complex between the benzophenone group of N27pBpa-cutinase and beta-CD was shown, and an apparent K-d of 1.65 mM was determined using H-1 NMR. Photocoupling of beta-CD to N27pBpa-cutinase in a 1:1 ratio, upon UVA irradiation at 360 +/- 20 nm, was shown by MALDI-TOF mass spectroscopy. UVA photoimmobilization of N27pBpa-cutinase on quartz slides coated with beta-CD was achieved from liquid or dry films by total internal reflection fluorescence (TIRF). The Dock'n'Flash method offers a solution for direct photocoupling and patterning of recombinant proteins onto surfaces with site-specific attachment,},
  author       = {Jensen, Rasmus L. and Stade, Lars W. and Wimmer, Reinhard and Stensballe, Allan and Duroux, Meg and Larsen, Kim L. and Wingren, Christer and Duroux, Laurent},
  issn         = {0743-7463},
  language     = {eng},
  number       = {13},
  pages        = {11597--11604},
  publisher    = {The American Chemical Society},
  series       = {Langmuir},
  title        = {Direct Site-Directed Photocoupling of Proteins onto Surfaces Coated with beta-Cyclodextrins},
  url          = {http://dx.doi.org/10.1021/la100950n},
  volume       = {26},
  year         = {2010},
}