Overexpression, rapid isolation, and biochemical characterization of Escherichia coli single-stranded DNA-binding protein.
Escherichia coli (E. coli) single-stranded binding protein (SSB) is a valuable protein for various biotechnical applications, such as PCR and DNA sequencing. Here we describe an efficient expression and purification scheme where the tendency of SSB to aggregate at low salt concentration and high protein concentration is avoided. The method contains fewer steps of purification and results in high protein yield, compared to previous published protocols. In our protocol, cells are harvested after cultivation overnight and SSB is isolated by ammonium sulfate precipitation followed by anion-exchange chromatography. The yield from a 2-liter fed-batch fermentor is 2 g protein, which is higher than all production methods for SSB earlier reported. Moreover, the two classical isolation steps combined in the purification scheme are robust, cost-efficient, and suitable for scaling up. The resulting SSB is pure and a correctly folded tetramer with an apparent binding to single-stranded DNA with a K(D) of 10(-8) M, as determined by surface plasmon resonance.
Ehn
M
M
Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm, Sweden.
Nilsson
P
P
Uhlén
M
M
Hober
S
S
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D001483:Base Sequence; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D020349:Surface Plasmon Resonance
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000645:Ammonium Sulfate