Revision as of 21:58, 16 December 2020

BstPol

Introduction

Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. This Bst polymerase I large fragment (BstPol) is a part of helimerase protein complex, which could be used with the aim to improve HDA method.

Biology

Since DNA polymerases are one of the main molecular biology tools, they are mostly used in different amplification methods such as PCR, RPA, LAMP or HDA^[1]. One type of polymerase, which can be used in these methods, is thermostable DNA polymerase I large fragment (EC: 2.7.7.7.), obtained from Bacillus stearothermophilus. This enzyme (67.7 kDa) has a strand displacement and 5’-3’ exonuclease activity. Based on scientific research, the best suitable polymerase activity temperature varies from 65 °C to 72 °C^[2].

In project FlavoFlow we a novel isothermal helicase dependent amplification method, which is based on DNA replication fork principle. Usually, in this assay, two complementary DNA strands are being separated with UvrD helicase. When newly generated ssDNA is being coated with single-stranded DNA-binding proteins. The next step of HDA process is the hybridization of site-specific primers to each ending border of ssDNA. After this hybridization the elongation process performed by DNA polymerase, starts^[1]. The biggest advantage of this HDA reaction is its specificity as well as the ability to perform the reaction at a constant temperature.

However, even if HDA method is sensitive and specific, it is able to amplify shorter than 200 bp sequences^[3]. This problem was solved by fusing TteUvrD helicase (BBa_K3416002) with Bst polymerase I large fragment (BstPol) through coiled-coil interaction (Fig. 1). Such fusion increases the speed and synthesis of new strands, which makes HDA reaction more specific and efficient. Based on similar researches, scientists have shown, that coordination between TteUvrD helicase and BstPol could increase specificity and efficiency of the HDA reaction^[3],^[4]. Previously mentioned proteins BstPol and TteUvrD are physically linked together by using coiled-coil structure. TteUvrD helicase is fused with one part of this structure, WinZip-A2 (WZA2), through the linker L1 and is possessed in the N-terminal end of the sequence. The research has shown that this type of coiled-coils structures provides predictable tertiary structure and stability^[5].

Figure 1. Bifunctional protein helimerase. TteUvrD - thermostable UvrD helicase, BstPol - Bst polymerase I large fragment, L1 - linker between WZB1/WZA2 coiled-coil amino acid structures, WZA2 - coiled-coil amino acid structure, fused with TteUvrD, WZB1 - coiled-coil amino acid structure, fused with BstPol, 10xHisTag - TteUvrD purification tag, Maltose binding protein (MBP) - TteUvrD purification tag, StrepII tag - BstPol purification tag.

The most important feature of these coiled-coil amino acid sequences is a seven-residue repeat (abcdefg)_n, where first (a) and fourth (d) positions need to be occupied by hydrophobic amino acids by the means to determine the oligomerization state. In parallel residues at seventh (g) and the succeeding fifth (e) positions forms hydrogen-bonded ionic interactions^[4]. In our case, these coiled coils were constructed based on c-Jun/c-Fos and GCN4 Leucine zippers, where parallel dimer is formed with the leucine in d sites and valine in a sites ^[3]. According to the research, the most stable heterodimer is formed with a T_M of 63 °C, where binding affinity constant reached Kd of 4.5 nM^[3].

Results

Helimerase – is a bifunctional protein complex, made up of two enzymes - BstPol and TteUvrD. These proteins are physically linked to each other through the coiled-coil structure. TteUvrD helicase is fused with one part of this structure, WinZip-A2 (WZA2), through the linker L1 and is possessed in the N-terminal end of the sequence. The research has shown that this type of coiled-coils structures provides predictable tertiary structure and stability^[3]. Firstly gene sequence WZA2-L1-TteUvrD was cloned into pETDuet vector. This protein was fused with a 10xHis tag and a maltose binding protein (MBP) at N terminus. Analogously, WZB1-L1-BstPol gene was cloned into the second MCS of pACYC plasmid and fused with StrepII tag at the N terminus.

The optimal expression conditions for WZA2-L1-TteUvrD and WZB1-L1-BstPol was observed in E. coli BL21(DE3) strain after 16 hours induction with 1 mM IPTG at 30 °C. Both proteins were purified under native conditions. WZA2-L1-TteUvrD protein was purified by using Ni-NTA affinity column. The concentration of recombinant protein observed after purification under native conditions from 1 L medium reached 5.5 mg/ml. However, as seen in SDS-PAGE gel, further optimizations procedures are needed to remove impurities. The yield of WZB1-L1-BstPol obtained after purification from 1 L medium was 2.2 mg/ml (Fig. 8).

Figure 8. SDS-PAGE electrophoresis after WZA2-L1-TteUvrD (136.598 kDa) and WZB1-L1-BstPol (74.225 kDa) purification. L - PageRuler Plus Prestained Protein Ladder (#26620), 1 - WZA2-L1-TteUvrD supernatant, 2 - WZA2-L1-TteUvrD 3 elution fraction, 3 - WZA2-L1-TteUvrD 5 elution fraction, 4 - WZA2-L1-TteUvrD 14 elution fraction, 5 - WZA2-L1-TteUvrD after protein concentration, 6 - WZB1-L1-BstPol supernatant, 7 - WZB1-L1-BstPol 4 elution fraction, 8 - WZB1-L1-BstPol 6 elution fraction, 9 - WZB1-L1-BstPol 7 elution fraction, 10 - WZB1-L1-BstPol after protein concentration. SDS-PAGE electrophoresis after WZA2-L1-TteUvrD (136.598 kDa) and WZB1-L1-BstPol (74.225 kDa) purification. L - PageRuler Plus Prestained Protein Ladder (#26620), 1 - WZA2-L1-TteUvrD supernatant, 2 - WZA2-L1-TteUvrD 3 elution fraction, 3 - WZA2-L1-TteUvrD 5 elution fraction, 4 - WZA2-L1-TteUvrD 14 elution fraction, 5 - WZA2-L1-TteUvrD after protein concentration, 6 - WZB1-L1-BstPol supernatant, 7 - WZB1-L1-BstPol 4 elution fraction, 8 - WZB1-L1-BstPol 6 elution fraction, 9 - WZB1-L1-BstPol 7 elution fraction, 10 - WZB1-L1-BstPol after protein concentration.

To verify that WZA2-L1-TteUvrD and WZB1-L1-BstPol proteins are stable at high temperatures we performed fluorescence thermal shift assay by utilizing a thermodynamic model3. This measurement is based on protein property to unfold upon increasing temperature of environment and solvatochromic property of fluorescence dye to fluorescent more intensively in the hydrophobic environment^[6]. The results (Fig. 9A and Fig. 9B) show that both proteins are stable at high temperatures, thus letting us use them in activity assays. Also, according to obtained melting temperatures (T_M) WZB1-L1-BstPol enzyme is more stable and starts to denature at 76 °C.

According to obtained enzymes stability, data kinetic experiments were performed. WZA2-L1-TteUvrD activity was measured based on SYBR GreenI fluorescent dye, which emits fluorescence upon binding with double-stranded DNA. Otherwise, if helicase separates dsDNA, when fluorescence declines proportionally1₀. As shown in Fig. 10A and Fig. 10B the bigger amount of WZA2-L1-TteUvrD protein is in the assay, the more dsDNA is unwinded.

WZB1-L1-BstPol activity (Fig. 11A and Fig. 11B) was also measured by observing its fluorescence depending on time. The bigger polymerase concentration is in the assay, the more amplified fragments are being generated. Linear dependence of polymerase activity on its substrate in solution was also determined by using fluorescence measurements.

Based on these enzymes activity assays we determined that the synthesis of active helimerase proteins was successful. However, after a lot of attempts we were unable to fuse these proteins via coiled-coil amino acid structures in vivo and in vitro. We hypothesized that newly formed affinity tags, as such as tertiary structures, could influence these proteins' interaction with each other, so in order to tackle this problem, new affinity tags and linkers should be tested. Also, with the aim to co-express these proteins in vivo, they could be cloned into other compatible plasmids.

Sequence and Features

References

1. ↑ ^{1.0 1.1} Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Reports 5, 795–800 (2004).
2. ↑ Kiefer, J. R. et al. Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 Å resolution. Structure 5, 95–108 (1997).
3. ↑ ^{3.0 3.1 3.2 3.3 3.4} Motré, A., Li, Y. & Kong, H. Enhancing helicase-dependent amplification by fusing the helicase with the DNA polymerase. Gene 420, 17–22 (2008).
4. ↑ ^{4.0 4.1} Muller, K.M., Arndt, K.M., Albert, T. Protein fusions to coiled-coil domains. Methods Enzymol. 328, 261-282.
5. ↑ Cimmperman, P. et al. A quantitative model of thermal stabilization and destabilization of proteins by ligands. Biophysical Journal 95, 3222–3231 (2008).
6. ↑ Cimmperman, P. et al. A quantitative model of thermal stabilization and destabilization of proteins by ligands. Biophysical Journal 95, 3222–3231 (2008).