Three single-point mutations were made in Bst DNA polymerase’s thumb domain at K549W, K582L, and Q584L (Fig. 1). Since this enzyme was designed for use in the LAMP portion of our point of care diagnostic test, stability was incredibly important. Outside of a laboratory setting, fluctuations in temperature ± 2 – 3°C can be expected, thus we aimed to increase the thermostability. Through these three mutations, we observed an improvement of Gibbs free energy from an initial 0 kcal/mol to -0.126, -1.63, and -2.039 kcal/mol at K549W, K582L, and Q584L, respectively.

Figure 1. Mutated residues on Bst DNA polymerase for improved enzyme thermostability. Thumb domain of the enzyme displayed in blue and altered amino acids in this region shown in orange.

Superimposition and sequence alignments of ϕ29 and Bst DNA polymerases indicated low structural similarity, which signified differing domain functions (Fig. 2). So, even with high enzyme processivity, any one of ϕ29 polymerase’s domains may not improve the function of our target enzyme.

Conversely, Bst is a structural homologue of Taq polymerase, with the one major difference being Bst’s lack of 5’ à 3’ exonuclease domain (Fig. 3). For this reason, previous research that successfully modified Taq in certain domains was a starting point for the protein fusion of our final enzyme.

Figure 2. Structural alignment of ϕ29 and Bst DNA polymerases. Low sequence and structural similarity seen between the enzymes. Bst shown in blue and ϕ29 in pink.

Figure 3. Structural alignment of Taq and Bst DNA polymerases. High sequence and structural similarity seen between the enzymes, excluding the 5’ à 3’ exonuclease domain in Taq. Bst shown in blue and Taq in green.

A (GGGGS)4 flexible linker was used to fuse Sso7d to Bst DNA polymerase, as to not interfere with either protein’s function (Fig. 4). In this sense, Sso7d acts like a clamp to hold the polymerase onto the DNA template, thereby improving its processivity and strand-displacing activity. This modified fusion enzyme was successfully expressed and purified.

Figure 4. Mutated Bst DNA polymerase fusion with DNA binding protein Sso7d. Flexible linker designed long enough to allow Sso7d to bind double-stranded DNA. Bst coloured in blue, (GGGGS)4 linker in purple, and Sso7d in yellow.

Our final enzyme is very similar to that of Figure 4, except for the fusion of a different DNA binding protein, Sac7e (Fig. 5). This protein is, however, a homologue of Sso7d, but with greater DNA binding affinity (1). Therefore, our fully modified Bst DNA polymerase is a significant improvement on previous research, as thermostability, processivity, and strand displacing activity are all enhanced.

Figure 5. Fully Modified Bst DNA polymerase via mutagenesis and protein fusion. 3-dimensional model of Bst with mutations at K549W, K582L, and Q584L, as well as fusion to Sso7d via a (GGGGS)4 linker displayed from front (a) and back (b) sides. Bst baseline structure displayed in blue, point mutations in orange, flexible linker in purple, and Sac7e in red.

All figures created using PyMOL modelling software.