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Study on Binding Affinity of a Glutathione S-Transferase (GST) Fusion Protein to DNA Probe

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DOI: 10.4236/abb.2015.65033    3,231 Downloads   3,634 Views  

ABSTRACT

The aims of this work were to: i) purify GST-fusion protein from bacterial cell extracts of Escherichia coli; ii) quantify the protein by SDS PAGE and Bradford assay; iii) determine protein-DNA interaction of the purified protein by Electrophoretic Mobility Shift Assay. Bacterial culture prepared by inoculation of a single E. coli colony that had a GST fusion protein (gst: six-X10 hd) constructed by ligation of the six-7-hd (X10) sequence into the BamHI and EcoRI sites of the vector pGEX-2T, grown overnight, was sonicated using Cole-Palmer Ultrasonic Homogenizer. Fusion protein was eluted from the beads with Tris-glutathione buffer (50 mM Tris [pH 8.1], 20 mM glutathione), which contained reduced Glutathione. SDS-PAGE was used to calculate the extracted bound protein. Total protein quantification was then estimated by the Bradford assay. Bovine Serum Albumin (BSA) absorbance values were used to plot the standard curve used to calculate the concentrations of the sample proteins. Nylon membrane was used for the electrophoretic transfer; membrane was cross linked and detected by Pierce’s Chemiluminescent Nucleic Acid Detection module. Results showed that X10 gave a strong band shift observed in Lanes 6 and 7 for both 200 ng and 400 ng elute 1 samples; however, there was no shift in the bands for the wild-type, positive control. The concentration of the elute 1 was obtained by the Bradford assay as 242.52 ng/μl and that of elute 2 was 106.30 ng/μl. Similarly, the result obtained by gel analysis was 300 ng/μl (0.3 μg/μl) and 150 ng/μl (0.15 μg/μl) for elutes 1 and 2 respectively.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Ali, F. and Hamza, N. (2015) Study on Binding Affinity of a Glutathione S-Transferase (GST) Fusion Protein to DNA Probe. Advances in Bioscience and Biotechnology, 6, 345-352. doi: 10.4236/abb.2015.65033.

References

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http://dx.doi.org/10.1093/nar/9.13.3047
[2] Guarente, L. and Bermingham-McDonogh, O. (1992) Conservation and Evolution of Transcriptional Mechanisms in Eukaryotes. Trends in Genetics, 8, 27-32.
http://dx.doi.org/10.1016/0168-9525(92)90021-U

  
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