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NUCLEOTIDES BINDING Class A β‐LACTAMASE FROM BACILLUS CEREUS 569/H/9
Author(s) -
Ott Taylor Ann,
Cha Peter W,
Shin Ben C,
Shaw Robert W.,
Kim SungKun
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.676.4
Subject(s) - systematic evolution of ligands by exponential enrichment , ethidium bromide , aptamer , bacillus cereus , nucleotide , biochemistry , cereus , chemistry , microbiology and biotechnology , biology , dna , rna , bacteria , genetics , gene
The Class A β‐lactamase from the pathogenic bacterium Bacillus cereus 569/H/9 catalyzes hydrolysis of β‐lactam antibiotics (e.g., Penicillin G), that impede the synthesis of bacterial cell wall. Although clavulanic acid is known as an inhibitor of Class A β‐lactamases, we are interested in the utility of aptamer inhibitors of Class A enzymes since they are such highly efficient and specific inhibitors of the Bacillus cereus 5/B/6 Class B enzyme. We have attempted to bind a random pool of nucleotides to the β‐lactamase using SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique. We found that the random pool of nucleotides bind β‐lactamase using the electrophoretic mobility shift assay (EMSA), confirmed by both ethidium bromide staining and coomassie blue staining. After 8 rounds of SELEX, we were able to proceed with inserting the selected nucleotides into a cloning vector in order to identify nucleotide sequences. We also carried out another method for selecting aptamers using the interaction between biotin and streptavidin. After 10 rounds of SELEX, we identified the sequences that bind the β‐lactamase. Using the computer program Mfold, the secondary structures of aptamers that potentially inhibit the β‐lactamase have also been determined.