Difference between revisions of "Part:BBa K4169000"

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Revision as of 15:05, 12 October 2022

Nattokinase gene with pelB

Signal peptide PelB with propeptide and mature nattokinase gene can produce active nattokinase in E. coli, and it is improved from the pelB leader sequence with aprN (without native signal peptide sequence)（https://parts.igem.org/Part:BBa_K4060021). Signal peptide gene of PelB can direct aprN to Escherichia coli periplasmic membrane. The propeptide has been proved to function as an intramolecular chaperone and is essential for guiding the correct folding of the linked protein to its mature form.

Usage and Biology

As for Signal peptide PelB with propeptide and mature nattokinase gene, there was no base sequence between PelB and nattokinase propeptide. At the same time, the base sequence of the mature peptide was significantly different, mainly in the last bit of the codon, but the amino acid sequence was basically unchanged, except that the 258th amino acid was changed from aspartic acid to serine.We finally succeeded in expressing active nattokinase as described in the experimental method described in reference^[1].

Nattokinase had potent fibrinolytic activity, which could be enhanced and prolonged in the plasma when it was taken orally. Based on its food origin and relatively strong fibrinolytic activity, nattokinase has advantages over other commercially used medicine in its preventative and prolonged effects, convenient oral administration, and stability in the gastrointestinal tract.

In our project, nattokinase was used to dissolve thrombus formed in blood vessels and prevent thrombus formation when thrombus is not formed. The mechanism of nattokinase has the following five points:
(1) Nattokinase activates the conversion of prourokinase to urokinase, together with tissue-type plasminogen to activate plasminogen to generate plasmin.
(2) Nattokinase promotes plasminogen transformation by stimulating vascular endothelial cells to produce tissue plasminogen activator (t-PA).
(3) Nattokinase also has the ability to degrade inactivated plasminogen activator type I inhibitor, promote the synthesis of more t-PA and urokinase, and indirectly promote thrombolysis.
(4) Nattokinase administration is capable of reducing the concentration of plasma coagulation factor VII and coagulation factor VIII that can increase the risk of cardiovascular diseases by triggering the coagulation cascade reaction.
(5) Nattokinase prevents the formation of thromboxane and significantly inhibits platelet aggregation mediated by collagen and thrombin, thereby delaying the formation of thrombi after oxidative arterial wall injury, and promoting the detachment of thrombi from blood vessel walls, so as to reduce endothelial damage and vascular endothelial thickening caused by thrombi^[2].

Structure

Mature peptides consist of 9 α-helixes, 9 β-sheets and 2 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.^[8]

Characterization

SDS-PAGE

First, we identified nattokinase by SDS-PAGE. We identified nattokinase produced by optimal expression conditions mentioned in the literature and it produced by our desired expression conditions.

Figure 1. SDS-PAGE analysis of nattokinase expression at 20 ℃+0.7mmol/L IPTG.

Through SDS-PAGE analysis, we found that there was no stripe in the supernatant, which may be because we didn’t use fermentation medium.Protein purity with Histag showed a clear stripe at 28kDa. At 40kDa there was only a shallow stripe, indicating the production of correct folded nattokinase rather than inclusion bodies.

Figure 1. SDS-PAGE analysis of nattokinase expression at 37 ℃+0.1mmol/L IPTG.

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 bodies at 37℃, therefore the concentration of nattokinase secreted into the supernatant was low. Protein purity with Histag showed a clear stripe at 28kDa. At 40kDa there was only a shallow stripe, indicating the production of correct folded nattokinase rather than inclusion bodies.

Bradford Assay

According to the literature, in order to get rich protein, the best incubation time is 20 hours, so in the early stages of the experiments, we use 20 hours as the incubation time. However, the protein gel map of the supernatant showed no ideal band, while the mature nattokinase band of 28kDa existed in the cells. We considered that IPTG may have failed due to long-term storage, the amount of protein secretion was too small to show the band on the protein gel. Therefore, we reconfigured 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 6 hours’ induction and determined the protein content. The results showed an increase in the amount of protein compared to the previous experiments.

Figure 3. Bradford analysis of protein concentration.

After that, we cultured the remaining bacterial solution continuously. After 20 hours of culture, the sample liquid was tested for protein content. Unexpectedly, the protein content has reduced.

Figure 4. Bradford analysis of protein concentration.

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 Bradford+IPTG and Bradford+LB medium, and found that the results were not due to the medium.

Figure 5. Bradford analysis of protein concentration.

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 out the best induction time of nattokinase in the 5mL system. In the future, we can connect nattokinase and fluorescent protein and determine the production amount of nattokinase 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 dropping onto fibrin plates for phenotypic testing.

Fibrin Plate

Figure 6. Bradford analysis of protein concentration.

In addition, we designed and performed the 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， at 37 ℃ of 0.1mmol/L, as the plate shows, a transparent circle. Moreover, the control group at the lower part of plate with 20μL water, no transparent circle was found.

Summary

We expressed mature nattokinase in E. coli.
We used Bradford Assay to find the optimal time for nattokinase production.
The nattokinase we expressed has the activity of solubilizing fibrin.

Reference

[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

Sequence and Features