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Expression and purification of putative Y‐family polymerase DinB from Sinorhizobium meliloti
Author(s) -
Kramer Caitlin,
DeLauteur Nicholas,
Brewer Tess,
Jones Kathryn,
Beuning Penny
Publication year - 2015
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.29.1_supplement.561.4
Subject(s) - sinorhizobium meliloti , biology , gene , genome , genetics , plasmid , escherichia coli , microbiology and biotechnology , mutant
The conserved Y‐family polymerase DinB is involved in translesion synthesis (TLS) of damaged DNA. The beneficial nitrogen‐fixing bacterium Sinorhizobium meliloti is a symbiont of leguminous plants. Because TLS polymerases enhance tolerance to stress, it is of interest to characterize these genes from S. meliloti, which has to survive an oxidative burst in order to establish the symbiosis. Genome annotation of S. meliloti indicates the presence of dinB genes on the endosymbiotic plasmid pSymA, which contains genes largely implicated in stress response, colonization, and nodulation, as well as on the chromosome. We cloned both putative dinB genes for protein expression and characterization. The pSymA DinB protein was overexpressed in Escherichia coli Rosetta pLysS cells with an N‐terminal 6His tag using the pET system, and purified by affinity chromatography. Characterization of the protein is ongoing. This work is supported by a Matz Scholarship and the American Cancer Society.