Header

UZH-Logo

Maintenance Infos

Knowledge-based design of reagentless fluorescent biosensors from a designed ankyrin repeat protein


Brient-Litzler, E; Plückthun, A; Bedouelle, H (2010). Knowledge-based design of reagentless fluorescent biosensors from a designed ankyrin repeat protein. Protein Engineering Design and Selection : PEDS, 23(4):229-241.

Abstract

Designed ankyrin repeat proteins (DARPins) can be selected from combinatorial libraries to bind any target antigen. They show high levels of recombinant expression, solubility and stability, and contain no cysteine residue. The possibility of obtaining, from any DARPin and at high yields, fluorescent conjugates which respond to the binding of the antigen by a variation of fluorescence, would have numerous applications in micro- and nano-analytical sciences. This possibility was explored with Off7, a DARPin directed against the maltose binding protein (MalE) from Escherichia coli, with known crystal structure of the complex. Eight residues of Off7, whose solvent accessible surface area varies on association with the antigen but which are not in direct contact with the antigen, were individually mutated into cysteine and then chemically coupled with a fluorophore. The conjugates were ranked according to their relative sensitivities. All of them showed an increase in their fluorescence intensity on antigen binding by >1.7-fold. The best conjugate retained the same affinity as the parental DARPin. Its signal increased linearly and specifically with the concentration of antigen, up to 15-fold in buffer and 3-fold in serum when fully saturated, the difference being mainly due to the absorption of light by serum. Its lower limit of detection was equal to 0.3 nM with a standard spectrofluorometer. Titrations with potassium iodide indicated that the fluorescence variation was due to a shielding of the fluorescent group from the solvent by the antigen. These results suggest rules for the design of reagentless fluorescent biosensors from any DARPin.

Abstract

Designed ankyrin repeat proteins (DARPins) can be selected from combinatorial libraries to bind any target antigen. They show high levels of recombinant expression, solubility and stability, and contain no cysteine residue. The possibility of obtaining, from any DARPin and at high yields, fluorescent conjugates which respond to the binding of the antigen by a variation of fluorescence, would have numerous applications in micro- and nano-analytical sciences. This possibility was explored with Off7, a DARPin directed against the maltose binding protein (MalE) from Escherichia coli, with known crystal structure of the complex. Eight residues of Off7, whose solvent accessible surface area varies on association with the antigen but which are not in direct contact with the antigen, were individually mutated into cysteine and then chemically coupled with a fluorophore. The conjugates were ranked according to their relative sensitivities. All of them showed an increase in their fluorescence intensity on antigen binding by >1.7-fold. The best conjugate retained the same affinity as the parental DARPin. Its signal increased linearly and specifically with the concentration of antigen, up to 15-fold in buffer and 3-fold in serum when fully saturated, the difference being mainly due to the absorption of light by serum. Its lower limit of detection was equal to 0.3 nM with a standard spectrofluorometer. Titrations with potassium iodide indicated that the fluorescence variation was due to a shielding of the fluorescent group from the solvent by the antigen. These results suggest rules for the design of reagentless fluorescent biosensors from any DARPin.

Statistics

Citations

11 citations in Web of Science®
11 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

1 download since deposited on 21 Jan 2011
0 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2010
Deposited On:21 Jan 2011 10:19
Last Modified:07 Dec 2017 04:45
Publisher:Oxford University Press
ISSN:1741-0126
Publisher DOI:https://doi.org/10.1093/protein/gzp074
PubMed ID:19945965

Download