Nattokinase gene with pelB
Nattokinase gene with pelB can produce active nattokinase in E. coli, and it is improved from the pelB leader sequence with aprN (without native signal peptide sequence)（BBa_K4060021 ). The signal peptide gene of PelB can direct nattokinase gene to the periplasmic membrane of E. coli. The propeptide of nattokinase has been shown to function as an intramolecular chaperone, and is essential for guiding the correct folding of the linked protein into its mature form.
Usage and Biology
As for the nattokinase gene with pelB, there is no sequence between pelB and nattokinase propeptide. At the same time, the sequence of the mature nattokinase is significantly different, mainly in the last bit of the codon, but the amino acids are basically unchanged, except for the 258th amino acid, which is changed from aspartic acid to serine. Moreover, unlike the pelB leader sequence with aprN, we finally succeeded in expressing the active nattokinase as described in the experimental approach described in ref[1].
Nattokinase has potent fibrinolytic activity, which can be enhanced and prolonged in plasma when taken orally. Based on its food origin and relatively strong fibrinolytic activity, nattokinase has advantages over other commercially available drugs in terms of its preventative and long-lasting effects, convenient oral administration, and stability in the gastrointestinal tract.In our project, we use nattokinase to dissolve thrombus formed in blood vessels and prevent thrombus formation when thrombus is not formed.
Structure
Mature nattokinase consists of nine α-helixes, nine β-sheets and two Ca2+ binding sites for structural stability. The catalytically active center of nattokinase consists of a conserved catalytic triad (Asp32, His64, Ser221), while its substrate-binding center contains three conserved amino acids (Ser125, Leu126, Gly127). The propeptide of nattokinase is involved in the correct folding of nattokinase as an intramolecular chaperone. The signal peptide gene of PelB can direct aprN to Escherichia coli periplasmic membrane and help mature nattokinase secretory expression.[2]
Characterization
SDS-PAGE
First, we identified nattokinase by SDS-PAGE. We identify the nattokinase produced by the optimal expression condition mentioned in ref. and the nattokinase produced by our desired expression condition.
From the SDS-PAGE analysis, we found no stripe in the supernatant, which may be due to the fact that we did not use a fermenting medium. Protein purity with Histag shows a clear stripe at 28kDa. At 40kDa there is only a shallow stripe, indicating the production of the correct folded nattokinase rather than the inclusion body(Figure 1.).
Through SDS-PAGE analysis, we found that there was no stripe in the supernatant, which may be because we reduced the induced concentration of IPTG in order to product correct folded nattokinase rather than inclusion body at 37℃ and did not use a fermenting medium, therefore the concentration of nattokinase secreted into the supernatant was low. Protein purity with Histag shows a clear stripe at 28kDa. At 40kDa there is only a shallow stripe, indicating the production of the correct folded nattokinase rather than the inclusion body(Figure 2.).
Bradford Assay
According to the literature, the optimal incubation time is 20 hours in order to obtain rich proteins, so in the early stages of the experiment we used 20 hours as the incubation time. However, the protein gel map of the supernatant did not show the desired stripe, while the mature nattokinase stripe of 28kDa is present in the cell. We considered that IPTG may have failed due to long-term storage and the amount of protein secreted is too small to show stripes on the protein gel. Therefore, we reconfigured the IPTG. At the same time, in order to test whether the failure of the previous experiment was caused by the IPTG, we took samples after six hours of induction and determined the protein content. The results showed an increase in the amount of protein compared to the previous experiments(Figure 3.).
After that, we continuously cultured the remaining bacterial solution. After 20 hours of culture, we tested the sample liquid for protein content. Unexpectedly, the protein content decreased(Figure 4.).
In order to test if the protein in LB culture medium influenced the content of protein in samples, we did a protein content test of IPTG with Bradford and LB medium with Bradford, and found that the results were not due to the medium(Figure 5.).
Therefore, we hypothesized that the volume of our culture system (5mL) was too small, leading to the depletion of nitrogen source in LB medium, then the exogenous protein nattokinase secreted by itself was used as nitrogen source for its own cell growth, resulting in a decrease in protein content in the supernatant. Due to the limited time, we did not find an optimal induction time for nattokinase in the 5mL regime. In the future, we can link the nattokinase gene with fluorescent protein and determine the amount of nattokinase production through the expression of fluorescent protein, so as to intuitively see the best induction time of nattokinase. The subsequent induction time was set at 6 hours for sampling and dropped onto a fibrin plate for phenotyping.
Fibrin Plate
In addition, we designed and performed an assay for the fibrinolytic activity of nattokinase. According to the literature, nattokinase has fibrinolysis activity [2], therefore, we used a fibrin plate to test nattokinase activity. The two holes at the upper part of the plate were added 20 μL supernantant and microbial respectively, which induced at 37 ℃ of 0.1mmol/L IPTG, as the plate shows, a transparent circle. Moreover, the control group with 20μL water at the lower part of plate, no transparent circle was found. This indicates that our expressed nattokinase is active and achieves the therapeutic purpose of our project.(Figure 6.).
[1] Han H M , Yong-Mei L I , University B , et al. Secretory Expression of Nattokinase from Bacillus natto in Escherichia coli[J]. China Biotechnology, 2014.
[2] Yuan L, Liangqi C, Xiyu T, Jinyao L. Biotechnology, Bioengineering and Applications of Bacillus Nattokinase. Biomolecules[J]. 2022 Jul 13;12(7):980