Direct site-directed photocoupling of proteins onto surfaces coated with beta-cyclodextrins.
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 molecular 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 (1)H 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.
Jensen
Rasmus L
RL
Department of Physics and Nanotechnology, Aalborg University, Skjernvej 4a, DK-9220 Aalborg, Denmark.
Städe
Lars W
LW
Wimmer
Reinhard
R
Stensballe
Allan
A
Duroux
Meg
M
Larsen
Kim L
KL
Wingren
Christer
C
Duroux
Laurent
L
D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D010777:Photochemistry; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D047392:beta-Cyclodextrins / Q000737:chemistry*
D011506:Proteins; D047392:beta-Cyclodextrins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase