Recombinant production of Thermus aquaticus single-strand binding protein for usage as PCR enhancer

Abstract

Single-stranded DNA-binding (SSB) proteins play an important role in DNA metabolism involving DNA replication, recombination, and repair in all living beings. In molecular biology, SSB proteins are used as enhancers to increase the efficiency and specificity of PCR. Thermostable SSB protein eliminates secondary structure or dimer formation and significantly increase the effectiveness of amplification of DNA fragments. In this study, it was ensured that the SSB gene of thermophilic bacteria Thermus aquaticus (T. aquaticus) was cloned into the pET28b vector and expressed in E. coli BL21 (DE3) PLysE cells. Then, the purification of the SSB protein produced in E. coli BL21 (DE3) PLysE cells was performed. 20 mg SSB protein was obtained from 1L bacterial culture, and its purity was more than 90%. It was shown by the PCR experiment that the SSB protein produced in this study could increase the amplification efficiency.

Keywords

• Polymerase Chain Reaction
• Single Strand Binding Protein
• Thermus aquaticus

References

1. 1. Csako, G., Present and future of rapid and/or high-throughput methods for nucleic acid testing. Clinica Chimica Acta, 2006. 363(1-2): p. 6-31.
2. 2. Ralser, M., et al., An efficient and economic enhancer mix for PCR. Biochemical and Biophysical Research Communications, 2006. 347(3): p. 747-751.
3. 3. Sahdev, S., et al., Amplification of GC-rich genes by following a combination strategy of primer design, enhancers and modified PCR cycle conditions. Molecular and Cellular Probes, 2007. 21(4): p. 303-307.
4. 4. Chen, X.Q., et al., Betaine improves LA-PCR amplification. Sheng Wu Gong Cheng Xue Bao, 2004. 20(5): p. 715-718.
5. 5. Spiess, A.N., N. Mueller, and R. Ivell, Trehalose is a potent PCR enhancer: lowering of DNA melting temperature and thermal stabilization of taq polymerase by the disaccharide trehalose. Clinical Chemistry, 2004. 50(7): p. 1256-1259.
6. 6. Schnoor, M., et al., Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer. Biochemical and Biophysical Research Communications, 2004. 322(3): p. 867-872.
7. 7. Kinoshita, E., E. Kinoshita-Kikuta, and T. Koike, A heteroduplex-preferential Tm depressor for the specificity-enhanced DNA polymerase chain reactions. Analytical Biochemistry, 2005. 337(1): p. 154-160.
8. 8. Dabrowski, S. and J. Kur, Cloning, overexpression, and purification of the recombinant His-tagged SSB protein of Escherichia coli and use in polymerase chain reaction amplification. Protein Expression and Purification, 1999. 16(1): p. 96-102.

9. 9. Cadman, C.J. and P. McGlynn, PriA helicase and SSB interact physically and functionally. Nucleic Acids Research, 2004. 32(21): p. 6378-6387.
10. 10. Genschel, J., U. Curth, and C. Urbanke, Interaction of E. coli single-stranded DNA binding protein (SSB) with exonuclease I. The carboxy-terminus of SSB is the recognition site for the nuclease. Journal of Biological Chemistry, 2000. 381(3): p. 183-192.
11. 11. Olszewski, M., et al., Application of SSB-like protein from Thermus aquaticus in multiplex PCR of human Y-STR markers identification. Molecular and Cellular Probes, 2005. 19(3): p. 203-205.
12. 12. Dabrowski, S., et al., Novel thermostable ssDNA-binding proteins from Thermus thermophilus and T. aquaticus-expression and purification. Protein Expression and Purification, 2002. 26(1): p. 131-138.
13. 13. Filipkowski, P., A. Duraj-Thatte, and J. Kur, Novel thermostable single-stranded DNA-binding protein (SSB) from Deinococcus geothermalis. Archives of Microbiology, 2006. 186(2): p. 129-137.
14. 14. Filipkowski, P., A. Duraj-Thatte, and J. Kur, Identification, cloning, expression, and characterization of a highly thermostable single-stranded-DNA-binding protein (SSB) from Deinococcus murrayi. Protein Expression and Purification, 2007. 53(1): p. 201-208.
15. 15. Filipkowski, P., M. Koziatek, and J. Kur, A highly thermostable, homodimeric single-stranded DNA-binding protein from Deinococcus radiopugnans. Extremophiles, 2006. 10(6): p. 607-614.
16. 16. Olszewski, M., et al., Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana. BMC Microbiology, 2010. 10: 260.
17. 17. Olszewski, M., M. Mickiewicz, and J. Kur, Two highly thermostable paralogous single-stranded DNA-binding proteins from Thermoanaerobacter tengcongensis. Archives of Microbiology, 2008. 190(1): p. 79-87.
18. 18. Bernstein D.A., et al., Crystal structure of the Deinococcus radiodurans single-stranded DNA-binding protein suggests a mechanism for coping with DNA damage. Proceedings of the National Academy of Sciences, 2004. 101(23): p. 8575-8580.
19. 19. Filipkowski P. and J. Kur, Identification and properties of the Deinococcus grandis and Deinococcus proteolyticus single-stranded DNA binding proteins (SSB). Acta Biochimica Polonica, 2007. 54(1): p. 79-87.
20. 20. Wadsworth R.I. and M.F. White, Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus. Nucleic Acids Research, 2001. 29(4): p. 914-920.
21. 21. Witte, G., R. Fedorov, and U. Curth, Biophysical analysis of Thermus aquaticus single-stranded DNA binding protein. Biophysical Journal, 2008. 94(6): p. 2269-2279.
22. 22. Jedrzejczak, R., et al., Structure of the single-stranded DNA-binding protein SSB from Thermus aquaticus. Acta Crystallographica Section D, 2006. 62(11): p. 1407-1412.
23. 23. Vallejo, L.F. and U. Rinas, Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microbial Cell Factories, 2004. 3 (1): 11.
24. 24. Kuduğ, H., et al., Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization. International Advanced Researches and Engineering Journal, 2019. 3(1): p. 20-25.
25. 25. Perales, C., et al., Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric single-stranded DNA-binding protein. Nucleic Acids Research, 2003. 31(22): p. 6473-6480.