Protocol abstract
Nature Protocols 4, - 893 - 901 (2009)
Published online: 21 May 2009 | doi:10.1038/nprot.2009.60
Subject Categories: Biochemistry and protein analysis | Genomics and proteomics | Nucleic acid based molecular biology
Construction and flow cytometric screening of targeted enzyme libraries
Navin Varadarajan1,2, Jason R Cantor2, George Georgiou1,2 & Brent L Iverson1,3
Abstract
Herein, we describe a methodology for the construction of targeted libraries intended to modify the substrate specificity of proteases expressed on the cell surface of Escherichia coli. The native outer membrane protease, OmpT, is used as a model system. The protocol relies on gene assembly using oligonucleotides and is easily adaptable to any enzyme in which information is available on the putative active site residues. Increasingly complex libraries can be generated in a systematic manner and screened using flow cytometry (fluorescence-activated cell sorting, FACS) for variants displaying altered function. Furthermore, if the substrate-binding pockets have not been elucidated, a protocol for partial multi-site saturation library construction is presented that allows for sampling a large number of residues, while maintaining an appropriate level of protein function. The entire procedure, from start to finish, should take approximately 2–3 weeks.
- Institute for Cell and Molecular Biology, University of Texas, Austin, Texas, USA.
- Department of Chemical Engineering, University of Texas, Austin, Texas, USA.
- Department of Chemistry and Biochemistry, University of Texas, Austin, Texas, USA.
Correspondence to: Brent L Iverson1,3 e-mail: biverson@mail.utexas.edu
Correspondence to: George Georgiou1,2 e-mail: gg@che.utexas.edu
nature-products
A-Z product listing
- 100 mm
15 mm Petri dishes(VWR) - 12 75 mm sterile culture tubes(VWR)
- Agarose(Sigma)
- Ampicillin(EMD Biosciences)
- BD Falcon Biodish XL(Becton Dickinson)
- BL21(DE3)(Invitrogen)
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