Direct Site-Directed Photocoupling of Proteins onto Surfaces Coated with beta-Cyclodextrins
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1629022
- author
- Jensen, Rasmus L. ; Stade, Lars W. ; Wimmer, Reinhard ; Stensballe, Allan ; Duroux, Meg ; Larsen, Kim L. ; Wingren, Christer LU and Duroux, Laurent
- organization
- publishing date
- 2010
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Langmuir
- volume
- 26
- issue
- 13
- pages
- 11597 - 11604
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- wos:000279239900163
- scopus:77954274027
- pmid:20441154
- 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
- 2016-04-01 09:48:48
- date last changed
- 2022-01-25 08:58:54
@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 (ACS)}},
series = {{Langmuir}},
title = {{Direct Site-Directed Photocoupling of Proteins onto Surfaces Coated with beta-Cyclodextrins}},
url = {{http://dx.doi.org/10.1021/la100950n}},
doi = {{10.1021/la100950n}},
volume = {{26}},
year = {{2010}},
}