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Nucleic Acids Research Advance Access originally published online on June 17, 2008
Nucleic Acids Research 2008 36(13):e78; doi:10.1093/nar/gkn363
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Nucleic Acids Research, 2008, Vol. 36, No. 13 e78
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Linking the functions of unrelated proteins using a novel directed evolution domain insertion method

Wayne R. Edwards1,2, Kathy Busse1, Rudolf K. Allemann2 and D. Dafydd Jones1,*

1School of Biosciences and 2School of Chemistry, Cardiff University, Cardiff, UK

*To whom correspondence should be addressed. Tel: +44 (0) 29 2087 4290; Fax: +44 (0) 29 2087 4116; Email: jonesdd{at}cf.ac.uk

Received April 11, 2008. Revised April 29, 2008. Accepted May 22, 2008.

We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the separate protein activities, thus providing a general strategy for constructing molecular switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 β-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b562 with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were observed in loops, several also occurred close to the termini of {alpha}-helices and β-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains.

Present address: Kathy Busse, Institut für Biochemie der Med. Fakultät, Universität Leipzig, Leipzig, Germany


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