Part:BBa_K4347011

Bst fusion with Sac7e and point mutations for enhanced thermal stability codon optimized for E.coli

This fusion protien was designed by linking the N-terminus of a modified Bst polymerase with thermostable DNA binding protien Sac7e using a flexible (GGGGS)₄ linker to increase polymerase thermostability and processivity in LAMP reaction.

Usage and Biology

Usage of bst and Sac7e in biology. Can summarize what was written for "bst with point mutations" and "sac7e" pages. This bst version DOES include the point mutations

A more thermally stable and processive polymerase

This final iteration of the new polymerase is an improvement of our previous part; BBa_K4347010, as it is a combination of our more thermally stable polymerase (BBa_K4347007) fused with DNA binding protien Sac7e (BBa_K4347006). The modified Bst polymerase contains three point mutations in the polymerase thumb domain: K549W, K582L and Q584L, which have been proven to improve thermal stability in Bst homologue Taq polymerase^[1]. The overall change in Gibbs free energy of wild-type Bst was calculated to be -150.13 kcal/mol, and the overall stability of the mutated Bst was calculated to be -152.03 kcal/mol thus indicative of a more thermally stable protein.

Thermal stability of Bst fusion protien computed from YASARA.

Along with an increased thermal stability, the mutated polymerase was fused to a DNA binding protien Sac7e to increase polymerase processivity during the LAMP reaction. Sac7e is isolated from thermoacidophilic archaeon Sulfolobus acidocaldarius and is part of the 7 kDa DNA-binding family^[2]. Sac7e binds to DNA without a strong sequence preference. In complex with DNA, a small beta-barrel is capped by anamphiphilic C-terminal a-helix. The triple-stranded beta-sheet is placed across the DNA minor groove with the intercalation of the Val26 and Met29 side-chains into DNA base-pairs, causing a sharp kink in the DNA duplex^[3]. 7 kDa DNA-binging protiens have been shown to increase processivity when fused to polymerases such as Taq^[4].

Fully modified Bst polymerase with fusion protien and point mutations modelled in Pymol.

Lab results and pictures

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 5
  Illegal XhoI site found at 209
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1015
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Xi, L. (2009, December 23). WO2009155464A2 - mutated and chemically modified thermally stable DNA polymerases. Google Patents. Retrieved July 12, 2022, from https://patents.google.com/patent/WO2009155464A2/en

2. Kalichuk, V., Béhar, G., Renodon-Cornière, A., Danovski, G., Obal, G., Barbet, J., Mouratou, B., & Pecorari, F. (2016). The archaeal “7 KDA DNA-binding” proteins: Extended characterization of an old gifted family. Scientific Reports, 6(1). https://doi.org/10.1038/srep37274

3. Su, S., Gao, Y.-G., Robinson, H., Liaw, Y.-C., Edmondson, S. P., Shriver, J. W., & Wang, A. H.-J. (2000). Crystal structures of the chromosomal proteins SSO7D/sac7d bound to DNA containing T-G mismatched base-pairs. Journal of Molecular Biology, 303(3), 395–403. https://doi.org/10.1006/jmbi.2000.4112

4. Wang, Y. (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Research, 32(3), 1197–1207. https://doi.org/10.1093/nar/gkh271