edoc

Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology

Mallin, Hendrik and Reuter, Raphael and Hestericova, Martina and Ward, Thomas R.. (2016) Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology. Nature Protocols, 11. pp. 835-852.

Full text not available from this repository.

Official URL: http://edoc.unibas.ch/53291/

Downloads: Statistics Overview

Abstract

Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot-p-L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-(N,N-diallylsulfamoyl)phenyl)-N,N,N-trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d.
Faculties and Departments:05 Faculty of Science > Departement Chemie
UniBasel Contributors:Ward, Thomas R. R. and Mallin, Hendrik and Reuter, Raphael and Ribar Hestericov√°, Martina
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Nature Publishing Group
ISSN:1754-2189
e-ISSN:1750-2799
Note:Publication type according to Uni Basel Research Database: Journal article
Identification Number:
Last Modified:25 Jan 2017 15:12
Deposited On:25 Jan 2017 15:12

Repository Staff Only: item control page