Revision as of 00:32, 19 October 2021

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].

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 WZB1-L1-BstPol was observed in E. coli BL21(DE3) strain after 16 hours induction with 1 mM IPTG at 30 °C. Protein was purified under native conditions. WZB1-L1-BstPol protein was purified by using Ni-NTA affinity column. The yield of WZB1-L1-BstPol obtained after purification from 1 L medium was 2.2 mg/ml (Fig. 2)

To verify that WZB1-L1-BstPol protein is stable at high temperature 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. The results (Fig. 3) show that protein is stable at high temperatures, thus letting us use it in activity assays. Also, according to obtained melting temperatures (T_M) WZB1-L1-BstPol enzyme is stable and starts to denature at 76 °C.

WZB1-L1-BstPol activity (Fig. 4A and Fig. 4B) 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.