Team:SZPT-CHINA/Project/Design
ABSTRACT
ABSTRACT
Food-derived antihypertensive peptides have good prospects for lowering blood pressure. Based on the existing antihypertensive peptide database, we designed new antihypertensive peptides with high activity using protein structure fingerprinting technology[1][2]. The ligation sequence of the antihypertensive peptide is designed according to the recognition site of the intestinal protease, so that it can be directly hydrolyzed by the intestinal hydrolase. Since the molecular weight of the antihypertensive peptide is small, we adopt a strategy of fusion expression. We designed a more convenient and effective way to lower blood pressure. It is through lactic acid bacteria that fusion protein containing antihypertensive peptide is directly produced in intestinal tract, and the protein can produce antihypertensive effect after being directly hydrolyzed and absorbed by intestinal hydrolase. Finally, to ensure safety，we designed a suicide mechanism regulated by glucose starvation.
Many scholars have conducted sufficient research on hypotensive peptides to confirm the positive role of antihypertensive peptides in lowering blood pressure[3][4]. In order to find antihypertensive peptides with higher activity, we designed new hypotensive peptides using protein structure fingerprinting technology. The specific design ideas are as follows:
- According to the existing antihypertensive peptide database, the first 10% of the experimental polypeptide of IC50 was screened;
- Further screening the high repetition rate polypeptide sequences supported by the literature as a reference;
- Analyzing the spatial structure of the reference sequence;
- Replace one of the amino acids but leave the original space unchanged;
- Re-retrieval to confirm the new hypotensive peptide sequence;
- In vitro activity assay was performed.
Antihypertensive Peptide Multimer
Since AHP consists of only a few amino acids, it is difficult to biosynthesize directly. So we selected five antihypertensive peptides and repeated in tandem to for mantihypertensive peptide multimer (AHPM).In this way ,we can get a lot of blood pressure lowering active peptides at one time. Two of them are our designed high-activity peptides KYLCY and FKGKYYP, and the other three are selected from the database which were verified with high-activity——VY, IPP and VPP.
How to Make the AHPM into Monomer Peptides with Hypotensive Activity?
We use arginine linkers (FR) to link AHPs according to the enzyme cleavage sites of common enzymes in the intestine——trypsin and α-chymotrypsin. Thus, such tandem AHPM can be recognized by the two enzymes and hydrolyzed into active antihypertensive peptides.
Fusion Expression
Although we have constructed a hypotensive peptide multimer, its molecular weight is still only about 10kDa, which is still difficult to biosynthesize in vivo.
Glutathione transferase (GST) is a commonly used fusion tag, so it is a good choice. In addition, it has been reported in the literature that amaranth seed globulin has surprising potential value for the conditioning of chronic diseases. And studies have been reported to insert bioactive peptides into its acidic subunits. Therefore, using amaranth seed globulin as a fusion label is better choice. However, we have also done two fusion expression strategies in different expression systems.
First, The fusion protein GST-AHMP was expressed in E. coli system. We inserted the hypotensive peptide multimer into the fusion expression vector pGEX-4T-2 and fused with glutathione transferase (GST) and expressed in E. coli BL21. The circuit design and production flow chart are as follows:
To make our products safer, the lactic acid bacteria expression syetem was choosed to express AHPM. First, we added the antihypertensive peptide multimer at the C-terminal of amaranth seed globulin to form fusion protein, which was then inserted into pET-28a (+)and expressed in E. coli BL21. The circuit is as follows：
Then we introduced AHPM-A11Sg into the lactic acid bacteria expression system. You can find out more on the ANTIHYPERTENSIVE PROBIOTICS page.
Lactococcus lactis, generally regarded as safe (GRAS), is a significant model lactic acid bacteria (LAB) without the potential of infection and pathogenicity. We chose Lactococcus lactis MG1363 to secrete A11Sg-AHPM directly in the human intestinal tract and then hydrolyze it into active antihypertensive peptide monomer by intestinal hydrolase (trypsin and chymotrypsin). In addition, we introduced a set of acidity sensors to regulate its expression.
We all know that antibiotic resistance has many potential hazards. In order to allow lactic acid bacteria to enter the human body directly without potential danger, we constructed its food-grade secretory expression vector. Nisin, encoded by the Nisin resistance gene nisI, is a lipoprotein produced by the nisin-producing bacterium to protect itself against the toxic effects of Nisin[5]. At the same time, nisin is also recognized as a safe natural preservative. Therefore, we replaced the erythromycin resistance gene in the lactic acid bacteria commonly used expression vector pMG36e with the Nisin resistance gene nisI. In order to allow our target protein to be secreted outside the cell without complicated separation and purification processes, we introduced a secretory signal peptide SPusp45[6].
Considering that the AHPM is easily degraded during the fermentation of the lactic acid bacteria or when it enters the gastric juice, we reconstituted a set of acidity sensors using the lactose operon of the lactic acid bacteria and the P170 system. The expression of lactic acid bacteria is inhibited when the pH is less than 5.5 in the environment, and when the pH is greater than 5.5 in the environment, the repression released and the target protein begins to express. The genetic circuit we designed is as follows:
When the pH in the environment is less than 5.5, the rcfB promoter senses changes of acidity and expresses the downstream repressor protein LacR. LacR protein binds to operons O1 and O2 to repress downstream gene expression[7].
However, when the pH in the environment is greater than 5.5, the rcfB promoter is inhibited. The LacR protein is degraded, and the repression is released.
To ensure that our products do not harm to humans and escape into the environment, we use the following strategies:
- Construction of food-grade expression vectors
- Gene derived from food-borne organisms
- Probiotics as a biological chassis
- Security Mechanism
Although we consider the safety of engineered bacteria from various sources such as genetic sources, biological chassis, and resistance, it is not unreasonable to be careful about biosafety. Therefore, it is very necessary to design a suicide mechanism that can be controlled.
We designed a safety mechanism based on the different substance between the human body and the environment. When probiotics are excreted in the feces, glucose is deficient in the environment, and glucose starvation induces the T-αcrp promoter to overexpress the autolytic enzyme gene (acmA)[8].
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