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Changing the recognition site of a conjugative relaxase by rational design

AuthorsGonzález-Pérez, Blanca; Carballeira, José Daniel; Moncalián, Gabriel ; Cruz, Fernando de la
Issue Date2009
CitationBiotechnology journal 4(4): 554-557 (2009)
AbstractTrwC is a relaxase protein, which starts and finishes DNA processing during bacterial conjugation in plasmid R388. TrwC recognizes a specific sequence of DNA (25 nucleotides) in the donor cell: the nic-site. As a model example, a single transversion C24G in nic avoids DNA processing by TrwC. Using this simple model, our objective was to obtain a proof of principle that TrwC specificity can be changed. Several structures of DNA-TrwC complexes were used as reference to design a focused saturation mutagenesis library (NNK) randomizing amino acid Lys262, since its side chain seems to sterically hinder the recognition of the C24G nic mutation by wild-type TrwC. Using bacterial conjugation as an in vivo selection system, several TrwC variants were found that show changes in substrate specificity. These variants were also tested in a competitive assay to evaluate their conjugation efficiency. © 2009 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim.
Identifiersdoi: 10.1002/biot.200800184
issn: 1860-6768
Appears in Collections:(IBBTEC) Artículos
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