Expression, purification and biochemical characterization of a single-stranded DNA binding protein from Herbaspirillum seropedicae

• Published in: Protein expression and purification Vol. 53; no. 1; pp. 195 - 200
• Main Authors: Vernal, Javier, Serpa, Viviane I., Tavares, Carolina, Souza, Emanuel M., Pedrosa, Fábio O., Terenzi, Hernán
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2007
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Chromatography, Gel; Cloning, Molecular; Cluster Analysis; Conserved Sequence; DNA binding; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Electrophoresis, Polyacrylamide Gel; Electrophoretic Mobility Shift Assay; Escherichia coli; Escherichia coli - genetics; Genome, Bacterial; Herbaspirillum; Herbaspirillum - genetics; Herbaspirillum seropedicae; Mass Spectrometry; Molecular Sequence Data; Molecular Weight; Open Reading Frames; Overexpression; Plasmids; Protein Binding; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Subunits - chemistry; Recombinant Proteins - biosynthesis; Sequence Homology, Amino Acid; SSB; Trypsin - pharmacology; SSB; Overexpression; DNA binding; Herbaspirillum
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2006.11.018

An open reading frame encoding a protein similar in size and sequence to the Escherichia coli single-stranded DNA binding protein (SSB protein) was identified in the Herbaspirillum seropedicae genome. This open reading frame was cloned into the expression plasmid pET14b. The SSB protein from H. seropedicae, named Hs_SSB, was overexpressed in E. coli strain BL21(DE3) and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein. The apparent molecular mass of the native Hs_SSB was estimated by gel filtration, suggesting that the native protein is a tetramer made up of four similar subunits. The purified protein binds to single-stranded DNA (ssDNA) in a similar manner to other SSB proteins. The production of this recombinant protein in good yield opens up the possibility of obtaining its 3D-structure and will help further investigations into DNA metabolism.