Expression, purification and biochemical characterization of a single-stranded DNA binding protein from Herbaspirillum seropedicae.
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.
Vernal
Javier
J
Laboratório de Expressão Gênica, Departamento de Bioquímica, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil.
Serpa
Viviane I
VI
Tavares
Carolina
C
Souza
Emanuel M
EM
Pedrosa
Fábio O
FO
Terenzi
Hernán
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D042803:Herbaspirillum / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010957:Plasmids; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000494:pharmacology
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D014357:Trypsin