APPROACHES FOR THE PURIFICATION OF E. COLI Β-CLAMP IN COMPLEX WITH THE C-TERMINAL DOMAIN OF THE Α-SUBUNIT OF DNA POLYMERASE III.

Abstract

Understanding the structural organization of replisome components is essential to elucidate the coordinated processes of DNA replication and repair in bacteria. This study designed to purify complexes formed between the α subunit (DnaE) of replicative polymerase the and the β-clamp (DnaN). To this end, DnaN and a 255-amino-acid C-terminal fragment of DnaE tagged with 6xHis and containing the iCBM consensus sequence (referred to as DnaE905hM) were individually overexpressed in E. coli B834 cells. While DnaN was found in the soluble fraction, DnaE905hM was localized to the insoluble fraction. The insoluble DnaE905hM was solubilized under denaturing conditions, bound to an affinity column, and refolded on-column in the presence of the β-clamp. The resulting complex was further purified using size-exclusion chromatography. Based on molecular weight predictions, a complex comprising one dimeric β-clamp (81.2 kDa) and one DnaE905hM subunit (26 kDa) was expected to elute around 180 ml, while a complex containing two DnaE905hM molecules was predicted to elute at approximately 175 ml. A prominent elution peak was observed at 173 ml, along with broader secondary peaks at 155, 205, and 220 ml. SDS-PAGE and Western blot analyses, using mouse anti-6x-Histidine antibodies conjugated with alkaline phosphatase and rabbit polyclonal anti-β-clamp antibodies, showed that β-clamp was most abundant in fraction 21, while DnaE905hM was primarily detected in fraction 22 likely representing their monomeric forms. Fainter bands of both proteins across fractions 15 to 19 suggest a range of complex stoichiometries.