Direct Site-Directed Photocoupling of Proteins onto Surfaces Coated with β-Cyclodextrins
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- 作者：Rasmus L. Jensen ; Lars W. Stde ; Reinhard Wimmer ; Allan Stensballe ; Meg Duroux ; Kim L. Larsen ; Christer Wingren ; Laurent Duroux
- 刊名：Langmuir
- 出版年：2010
- 出版时间：July 6, 2010
- 年：2010
- 卷：26
- 期：13
- 页码：11597-11604
- 全文大小：899K
- 年卷期：v.26,no.13(July 6, 2010)
- ISSN：1520-5827
文摘
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 β-cyclodextrin (β-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 β-CD was shown, and an apparent Kd of 1.65 mM was determined using 1H NMR. Photocoupling of β-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 β-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.