PEDS Advance Access published online on June 15, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm026
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Methods |
Avoiding and controlling double transformation artifacts
1 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel 2 B division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
3 To whom correspondence should be addressed. E-mail: tawfik{at}weizmann.ac.il
This article describes a set of standard control experiments for the authentication of new protein variants isolated through library selection and site-directed mutagenesis. These controls are specifically designed to rule out artifacts derived from ‘double transformants’—i.e. cells transformed with, or infected by, two different plasmids simultaneously. These seem to have been the source of past artifacts and, as demonstrated here, are far more common than generally recognized. By following standard protocols for cloning, plasmid isolation, subcloning, in combination with functional assays, the presence of such artifacts can be ruled out. This protocol needs to be applied for any new variant isolated from heterogeneous gene repertoires, and in particular for variants isolated by selection for either enzymatic activity, or binding.
Keywords: artifacts/directed evolution/double transformations/library selection/plasmid DNA
Received May 13, 2007; revised May 13, 2007; accepted May 14, 2007.
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