Team:SZPT-CHINA/Project/Result

RESULTS

DESIGN OF HIGHLY ACTIVE ANTIHYPERTENSIVE PEPTIDES

The Amino Acid Sequence of New Design Antihypertensive Peptides

Highly active antihypertensive peptides (AHPs) are needed first. 10 high-activity antihypertensive peptides were designed by protein structure fingerprinting software. Then the AHPs were synthesized and analyzed for in vitro activity. In vitro activity was evaluated by IC50, and the smaller the IC50, the higher the activity of blood pressure lowering. The amino acid sequence and in vitro activity IC50 of 10 AHPs are shown in Tab. 1. As shown in Fig. 1, only 6 AHPs have antihypertensive activity. And the KYLCY has the highest activity compared to the peptides in the current AHP database.

Fig.1 In Vitro Activity of AHPs

Construction of The Antihypertensive Peptides Multimer (AHPM)

Since AHPs are composed of only a few amino acids, it is difficult to directly biosynthesize. The newly designed high activity hypotensive peptides KYLCY and FKGKYYP and fully validated VY, IPP and VPP selected from the database were used to construct AHPM. The AHPM containing 78 amino acids was joined by FR, which is cleavage sites of common enzymes in the intestine, trypsin and α-chymotrypsin. The amino acid sequence of AHPM is shown in Fig.2.

Fig.2 Amino Acid Sequences of AHPM

FUSION EXPRESSION OF AHPM IN E. coli SYSTEM

Construction and Identification of Recombinant Expression Vector pGEX-4T-2-AHPM

The AHPM gene was cloned into the pGEX-4T-2 expression vector by EcoR Ⅰ and Sal Ⅰ restriction enzyme sites in the 5' and 3' regions, respectively. Positive clones from LB plates containing 100 μg/mL ampicillin were selected for restriction analysis and PCR. As shown in Fig.3, Lane 1 and lane 2 both have fragments of 246 bp, which is the same as AHPM. The result show that the recombinant expression vector named pGEX-4T-2-AHPM was constructed successfully.

Fig.3 Analysis of recombinant expression vector pGEX-4T-2-AHPM. Lane M: DNA marker. Lane 1: pGEX-4T-2-AHPM / EcoR Ⅰ and Sal Ⅰ; Lane 2: PCR of pGEX-4T-2-AHPM ;

Expression of GST-AHPM In E. coli BL21

The recombinant strain E. coli BL21(DE3) with pGEX-4T-2-AHPM vector was induced by IPTG for 6 hours and then collected for SDS-PAGE and Western blot analysis. The results of SDS-PAGE were shown in fig.4. After IPTG induction, the E. coli BL21 transferred with pGEX-4T-2 vector produced 26kDa GST protein (lane2) and E. coli BL21 transferred with pGEX-4T-2-AHPM produced 36kDa protein matched well with our GST-AHPM fusion protein (lane 4). In contrast, Non-induced E. coli BL21 cells did not express GST or GST-AHPM (lane 1, lane3). Western blot results showed that 26kDa GST protein and 36kDa fusion protein carry GST tag (Fig 4B). The results showed that the constructed tandem AHP could be expressed in the fusion expression system.

Fig.4 Analysis of GST-AHPM expression in E.coli BL21.

A.SDS-PAGE; B. Western blot ;Lane 1: E.coli BL21 without IPTG Inducing; Lane 2: E.coli BL21(pGEX-4T-2) with IPTG Inducing; Lane 3:E.coli BL21(pGEX-4T-2-AHP) without IPTG Inducing ; Lane 4: E.coli BL21(pGEX-4T-2-AHP) ) with IPTG Inducing.

In Vitro Activity of AHPM

The fusion protein GST-AHPM was purified by GST affinity chromatography. After trypsin and α-chymotrypsin digestion, the GST protein was removed by GST affinity filler and then the polypeptide mixture was obtained. The activity of AHP multimer was determined in vitro. The in vitro activity of the mixed peptide is shown in the Fig.5 The IC50 of inhibiting ACE activity is 0.1210mg/ml.

Fig.5 In Vitro Activity of AHPM

Preparation of AHPs Nanocapsules

In order to increase the sustained release performance and bioavailability of AHP. The AHPs obtained by hydrolysis were used as a core material, and PLGA was used as a wall material to prepare an antihypertensive peptide nanocapsule. As shown in the SEM image of Fig. 6, the particle size distribution of the microspheres is relatively evenly distributed between 300 and 1000 nm, which indicates the successful preparation of the antihypertensive peptide nanocapsules.

Fig.6 Scanning Electron Micrograph of AHPs Nanocapsules

EXPRESSION OF A11SG-AHPM IN LACTIC ACID BACTERIA

Construction of Yhe Recombinant Plasmid pET-28a(+)-A11Sg-AHPM

Because the GST protein is non-foodborne, A11S globulin seed storage protein (A11Sg) is selected for fusion expression with AHPM, and studies have shown that the hydrolysate of the protein also has hypotensive activity.

The A11Sg-AHPM gene was cloned into the pET-28a (+) expression vector, and the recombinant expression was named pET-28a (+)-A11Sg-AHPM. As shown in Fig.7, Lane 1 and lane 2 both have fragments of 1735 bp, which is the same as A11Sg-AHPM. The result show that the recombinant expression vector named pET-28a (+)-A11Sg-AHPM was constructed successfully.

Fig.7 Analysis of Recombinant Expression Vector pET-28a (+)-A11Sg-AHPM. M: DNA Marker; Lane 1: pET-28a (+)-A11Sg-AHPM /Xba I and BamH I; Lane2: PCR of pET-28a(+)-A11Sg-AHPM

Expression of A11Sg-AHPM in E. coli BL21

The recombinant pET-28a (+)-A11Sg-AHPM transferred to E.coli BL21(DE3) and the recombinant E.coli BL21(DE3) were selected on LB plates containing 50μg/mL Kanamycin. Then the cells were induced by IPTG and the results of protein electrophoresis were shown in Fig.8. The SDS-PAGE results showed E. coli BL21 transferred with pET-28a(+)-A11Sg-AHPM produced a 66kDa protein (lane 3) which was a little bigger than expect 64kDa fusion protein. Western blot results showed that the constructed A11Sg-AHPM protein could be expressed in E. coli.

Fig.8 Analysis of A11Sg-AHPM expression in E.coli BL21.

A.SDS-PAGE; M: Marker; Lane 1: E.coli BL21 without IPTG inducing; Lane 2: E.coli BL21 (pET-28a (+)-AHP without IPTG inducing; Lane 3: E.coli BL21 (pET-28a (+)) with IPTG inducing. B indicate Western blot analysis of A11Sg-AHPM

In Vitro Activity of A11Sg-AHPM

The fusion protein A11Sg-AHPM was purified by His affinity chromatography. The mixture of A11Sg and AHP peptides was obtained by trypsin hydrolysis. The in vitro activity of the mixture is shown in the Fig.9. The IC50 that inhibits the activity of ACE is 1.28mg/mL

Fig.9 In vitro activity of A11Sg-AHPM enzymatic mixture

Construction of Recombinant Plasmid pMG36e- A11Sg-AHPM

The verified A11Sg-AHPM was inserted into the LAB expression vector pMG36e. In order to allow the fusion protein to be secreted into the intestine, we also linked to the secretory signal peptide SPusp45. Furthermore, in order to us pH control promoter to regulate the expression of A11Sg -AHPM, we also inserted the O1/O2 operator which will be combined by LacR protein. So, the fragment O1/O2-SPusp45-A11Sg-AHPM (OOUAA) was synthesized and inserted in the pMG36e vector. The recombinant vector named pMG36e-A11Sg-AHPM and was confirmed by restriction analysis and PCR(Fig.10). The results showed the fragment with size 2124bp could be obtained by restricted (lane1) and amplified (lane2). The results suggested that the recombinant expression vector was constructed.

Fig.10 Analysis of Recombinant Expression Vector pMG36e- A11Sg-AHPM. M: DNA Marker; Lane 1: pMG36e- A11Sg-AHPM / EcoR Ⅰand Sal Ⅰ; Lane 2: PCR of pMG36e- A11Sg-AHPM

A11Sg-AHPM Protein Expression on Lactococcus Lactis MG1363

The pMG36e-A11Sg-AHPM recombinant vector transferred to Lactococcus Lactis MG1363（LAB MG1363） by Electroporation and cultured in GM17 medium. The fermentation broth was collected and detected by SDS-PAGE. The results of protein electrophoresis in Fig.11 indicated that the fusion protein can also be expressed in Lactococcus Lactis MG1363 and that the fusion protein exists in the fermentation broth, which means that SPusp45 successfully secreted the protein to the outside of the cell.

Fig.11 SDS-PAGE Analysis of A11Sg-AHPM Protein Expression in Lactococcus lactis MG1363.M: Marker; Lane 1: LAB MG1363 (pMG36e) soluble protein; Lane 2: LAB MG1363 (pMG36e-A11Sg-AHPM) soluble A11Sg-AHP; Lane 3: LAB MG1363 (pMG36e) insoluble protein; Lane 4: LAB MG1363 (pMG36e-A11Sg-AHPM) insoluble A11Sg-AHPM; Lane 5: LAB MG1363 (pMG36e-A11Sg-AHPM) fermentation supernatant without SPusp45; Lane 6: LAB MG1363 (pMG36e-A11Sg-AHPM) fermentation supernatant with SPusp45

In Vitro Activity of Recombinant Lactococcus Lactis MG1363

The collected fermentation supernatant of lactic acid bacteria was hydrolyzed with trypsin and α-chymotrypsin to determine its in vitro activity. Because we want to use lactic acid bacteria with antihypertensive function. The A11Sg-AHPM protein could not be purified. In a word, after fermented, the supernatant was hydrolyzed and analyzed activity directly. The LAB MG1363 transferred with pMG36e was as the control group. The results were showed in Fig.12. The IC50 of supernatant of LAB MG1363 transferred with pMG36e-A11Sg-AHPM was 4.8 mg/ml. Compare the control supernatant, the IC50 result indicated that the fermentation supernatant of LAB MG1363 transferred with pMG36e- A11Sg-AHPM has much higher inhibitor activity than control group. It shows that the fusion protein with antihypertensive peptide can be secreted outside the cell, and has a high blood pressure lowering activity after enzymatic hydrolysis.

Fig.12 In vitro activity of MG1363 fermentation supernatant

Analysis of PrcfB Promoter Regualtion In Lactococcus Lactis MG1363

From above result, we can confirm that the A11Sg-AHPM can be expressed in LAB MG1363. Then we inserted the PrcfB-LacR(P-L) gene in LAB MG1363 to prevent degradation of AHPM by lowing pH. Fig13 showed that (P-L) gene with the size of 1172bp was inserted in pMG36e-A11Sg-AHMP successfully (Fig.13). After we constructed the hydrogen ion regulation system, the recombinant bacteria were cultured in different pH media. The results are shown in Fig.14.There is almost no expression of fusion protein at pH < 5.5, and fusion protein expression is only available at pH>5.5. It indicates that the hydrogen ion regulation mechanism can play a role in the recombinant LAB MG1363.

Fig.13 Analysis of Recombinant Expression Vector pMG36e-A11Sg-AHPM-P-L. M: DNA Marker; Lane 1: pMG36e-A11Sg-AHPM-P-L / Hind III and Sal Ⅰ; Lane2: CR of pMG36e-A11Sg-AHPM-P-L

Fig.14 The Expression of A11Sg-AHPM in Different pH M: DNA Marker; Lane 1: LAB MG1363(pMG36e-A11Sg-AHPM-P-L) cultured in pH5; Lane 2: LAB MG1363(pMG36e-A11Sg-AHPM-P-L) cultured in pH5.5; Lane 3: LAB MG1363(pMG36e-A11Sg-AHPM-P-L )cultured in pH6; Lane4: LAB MG1363(pMG36e-A11Sg-AHPM-P-L) cultured in pH7

Expression of NisI In Lactococcus Lactis MG1363

Since the biological chassis is ultimately to be valued in the intestine, a food grade resistant expression vector is required. We replaced the erythromycin resistance gene of the original plasmid pMG36e with the nisin resistance gene nisI. As shown in Fig.15, the restriction and PCR result show that nisI with size 856bp was inserted in pMG36e successfully. The recombinant vector named pMG36N

Fig.15 Analysis of Recombinant Expression Vector pMG36N. M: DNA Marker ; Lane 1: pMG36N/EcoR Ⅰand Nhe Ⅰ;Lane 2: PCR of pMG36N

The LAB MG1363 transferred with pMG36N were spread on the plate with nisin and erythromycin respectively. The result is showed in Fig.16, we can see that the recombinant strain could grow on the nisin-resistant plate, could not grow on the Emr-resistant plate, indicating that the resistance gene replacement was successful.

Fig.16 The Growth of MG1363 on different resistance plate

The LAB MG1363, LAB MG1363 with pMG36e and LAB MG1363 with pMG36N were inoculated in GM17 medium with 0,10,20,30,40,50,60,70,80,90,100 IU/ml nisin respectively. As shown in Fig. 17, After 12 hours of culture, samples were taken and measured the OD600 to compare their growth.

Fig.17 Analysis of Nisl Resistance Verification

The result showed that the growth of LAB MG1363 and LAB MG1363 transferred with pMG36e was inhibited strongly in GM17 culture medium with nisin, while the LAB MG1363 transferred with pMG36N still growth very well. This demonstrated that the gene nisI encoding lipoprotein nisI is expressed in MG1363 and confers the resistance to nisin in this strain. But as the concentration of nisin increases, the growth of this strain is also depressed.

Analysis of Suicide Mechanism

To ensure that the transgenic bacteria are not harmful to the environment, we constructed the autolytic enzyme gene acmA of the lactic acid bacteria downstream of the glucose starvation promoter. The results of the construction of the recombinant plasmid pMG36e-Pα-Tcrp-acmA named pMG36e-P-a are shown in Fig. 18. The results of LAB MG1363 and LAB MG1363 with pMG36e-P-a after induction of recombinant bacteria by different glucose concentrations are shown in Fig. 19. The results showed that when the concentration of glucose in the environment was less than 0.05%, the growth of recombinant bacteria was significantly inhibited.

Fig.18 Analysis of Recombinant Expression Vector of pMG36e-P-a.M: Marker; Lane 1: pMG36e-P -a / EcoN I and Sph I; Lane 2: PCR of pMG36e-P -a

Fig .19 Analysis of Suicide Mechanism