Formation of Persister in Klebsiella pneumoniae: Recombinant Expression and Purification of hipA and hipB proteins from K. pneumoniae
Publication Date : 06/11/2015
Several bacterial populations areknownto be containing some fraction of cells which survive exposure to antibiotics and harsh environment, are called as persister cells. This fraction of cells is very small generally ranging from 10-7 to 10-5. The mechanism of persister formation is not yet clearly understood although expression of toxin-antitoxin (TA) pairs of proteins has been found to be associated with persister formation. Klebsiella pneumoniae is also shown to produce persister cells by prolonged exposure to ampicillin.In this study, we have identified a pair of proteins, hipA and hipB, of TA system in Klebsiella pneumoniae. The proteins have 70% and 60% sequence similarity respectively with their homologous proteins from E. coli. hipA and hipB associate together to regulate survival of persister cells by binding to DNA in unfavourable conditions. Both hipA and hipB proteins from Klebsiella pneumoniae were cloned, expressed and purified. The clones were over expressed in fusion with His-tag in E. coli strains BL-21 (DE3). The purification was done using Immobilized metal affinity chromatography having Ni-NTA matrix. Sequence analysis by in-silico methods shows that hipA and hipB from K. pneumoniae may have different pattern of oligomer formation and DNA recognition than their counterparts in E. coli.
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