Changing the recognition site of a conjugative relaxase by rational design.
作者： Blanca González-Pérez , José Daniel Carballeira , Gabriel Moncalián , Fernando de la Cruz
关键词:
摘要: TrwC is a relaxase protein, which starts and finishes DNA processing during bacterial conjugation in plasmid R388. recognizes specific sequence of (25 nucleotides) the donor cell: nic-site. As model example, single transversion C24G nic avoids by TrwC. Using this simple model, our objective was to obtain proof principle that specificity can be changed. Several structures DNA-TrwC complexes were used as reference design focused saturation mutagenesis library (NNK) randomizing amino acid Lys262, since its side chain seems sterically hinder recognition mutation wild-type an vivo selection system, several variants found show changes substrate specificity. These also tested competitive assay evaluate their efficiency.
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