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Complete labelling of pneumococcal DNA-binding proteins with seleno-L-methionine

AuthorsLorenzo-Díaz, Fabián ; Moreno-Córdoba, Inmaculada; Espinosa, Manuel
KeywordsStreptococcus pneumoniae
Protein purification
Protein labelling
DNA-protein interactions
Issue Date10-Sep-2019
CitationJournal of Microbiological Methods (2019)
AbstractStreptococcus pneumoniae is a pathogenic and opportunistic Gram-positive bacterium that is the leading cause of community-acquired respiratory diseases, varying from mild- to deathly- infections. The appearance of antibiotic-resistant isolates has prompted the search for novel strategies and targets to tackle the bacterial resistances. One of the most promising approaches is the structure-based knowledge of possible targets in conjunction with rational design and docking of inhibitors of the chosen targets. A useful technique that helps to solve protein structures is to label them with an amino acid derivative like seleno-methionine that facilitates tracing of some of the amino acid residues. We have chosen two pneumococcal DNA-binding proteins, namely the relaxase domain of MobM protein from plasmid pMV158, and the RelB-RelE antitoxin-toxin protein complex. Through several changes that improve substantially a previous protocol (Budisa et al., 1995), we have used seleno-L-methionine to incorporate selenium into the amino acid sequence of the selected proteins. We have achieved 100% labelling of the proteins and could demonstrate that the labelled proteins retained full activity as judged from the relaxation of supercoiled plasmid DNA and from gel-retardation assays.
Description27 p.-4 fig.-3 tab.
Publisher version (URL)https://doi.org/10.1016/j.mimet.2019.105720
Appears in Collections:(CIB) Artículos
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