Cloning and Biochemical Characterization of Human ssDNA Binding Proteins
Single strand binding (SSB) proteins play important roles in many aspects of cellular DNA metabolism. This include protecting the single stranded DNA (ssDNA) from nucleolytic digestion and preventing formation of undesired secondary structures in ssDNA, thus keeping it in an appropriate conformation for the action of enzymes involved in processes like DNA replication, repair and recombination. In addition to the major eukaryotic SSB-protein, human replication protein A (hRPA), humans cells contain multiple small nuclear ssDNA binding proteins, including hSSB1 which has a domain organization closer to that of the archeal SSB-protein. There is a multitude of studies focused on the biochemical features of hRPA, but hSSB1 and hSSB2 have been largely neglected. In order to investigate the biochemical mechanism of hSSB1 and hSSB2, the genes that encode for these proteins were cloned using PCR and electrophoresis techniques. After the purification of the amplified open reading frames, we constructed a vector by the inserting the genes into a plasmid using restriction exonuclease digestion and ligation. Then, E.coli cells were transformed using the constructed vector. When the successful construction of the desired expression plasmids was confirmed by sequencing, we transformed into the E.coli expression strains containing T7 RNA polymerase on DE3 lysogen. Protein expression was induced by adding IPTG into the medium. The proteins were extracted from the cells by lysis, purified using nikel column, heparin column and gel filtration column chromatography. Using fluorescence polarization anisotropy binding assay with a synthesized ssDNA, the SSBs binding affinity was verificated.
School:
University of Puerto Rico at Humacao
Department:
Industrial Chemistry
Research Advisor:
Maria Spies
Department of Research Advisor:
Biochemistry
Year of Publication:
2008