Research On Capitol Hill 2014
SSBs are DNA binding proteins that are essential components of cells and play key roles in DNA replication, repair, and recombination. Here we utilize two biochemical properties associated with the E. coli SSB protein to develop a novel procedure to purify proteins using a resin-free strategy to combat the largest bottleneck in biochemical research— obtaining uncontaminated, single proteins from the total cellular contents.
1. E. coli SSB binds to single stranded DNA (ssDNA) with extremely high affinity.
2. It is also a unique protein with respect to its purification—it is possible to obtain greater than 95% pure SSB from the total cell lysate without using any sort of column or resin, utilizing polyethyleneimine (PEI) and ammonium sulfate precipitation.
Our design uses SSB as an affinity/solubility tag to enhance the solubility and expression of difficult-to-purify proteins, and allows for the simple, resin-free purification using PEI and ammonium sulfate precipitation. Constructs have been made that include the SSB gene, along with the ability to fuse any gene of interest, as well as a TEV Protease cleavage sequence allowing for proteolytic cleavage after gene expression. Two genes of interest have been cloned in thus far— TEV protease and Rad51.
Soffe, Mark, "A Column Free Protein Purification Procedure using E. coli Single ‐ Stranded DNA Binding Protein (SSB) as an Affinity Tag" (2014). Research On Capitol Hill 2014. Research on the Hill (Salt Lake City). Paper 19.