Premium
Purification and characterization of a bacterial mating protein
Author(s) -
Bulku Artemisa,
Berkmen Melanie
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.574.9
Subject(s) - histidine , chemistry , biochemistry , recombinant dna , antibody , electrophoretic mobility shift assay , escherichia coli , chromatography , microbiology and biotechnology , biology , biophysics , enzyme , genetics , gene expression , gene
Conjugation is the transfer of DNA from a donor to a recipient bacterium through a DNA translocation channel known as a Type IV Secretion System (T4SS). Our work focuses on the T4SS of ICEBs1, the integrative and conjugative element found in Bacillus subtilis. ConE is a component of the ICEBs1 T4SS. ConE is a VirB4‐like ATPase, most likely utilizing the energy generated from ATP hydrolysis for DNA transfer or T4SS assembly. Previously, we fused a hexa‐histidine tag (His6) to ConE for ease of purification via nickel affinity chromatography. However, we found that surprisingly low concentrations of imidazole inadvertently wash His6‐ConE off the nickel resin, preventing us from obtaining a high yield of pure protein. Here, we cloned deca‐histidine tagged ConE (His10‐ConE) and purified it side by side with His6‐ConE. We observed that His10‐ConE can be purified in higher yield and with fewer contaminants than His6‐ConE. After optimization, we conducted a large‐scale purification of His10‐ConE for the purpose of obtaining antigen for production of antibodies to ConE. In the future, the antibodies will be used in western blots, pull‐down assays, and immunofluorescence microscopy to characterize ConE levels, interactions, and localization, respectively. Additionally, we plan on exploring His10‐ConE's ability to interact with DNA using electrophoretic mobility shift assays.