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According to the results, we can see that the E.coli recombinant expression system is a good way to produce AMPs because of its easy culture, fast growth and larger quantities than those purified from their natural sources. What’s more, this system costs less money compared with chemical synthesis.
According to the results, we can see that the E.coli recombinant expression system is a good way to produce AMPs because of its easy culture, fast growth and larger quantities than those purified from their natural sources. What’s more, this system costs less money compared with chemical synthesis.
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To detect the antimicrobial activity of the recombinant CecropinXJ, we did the inhibition zone assay towards Staphylococcus aureus (as well as Bacillus subtilis, Klebsiella pnuemoniae, Escherichia coli, Enterococcus faecalis, Microsporum canis and Trichophyton rubrum with the assistance of TMMU_China) and detected the growth situation of them after recombinant CecropinXJ was added into the bacterial culture solution. To obtain recombinant CecropinXJ solution, 10ml culture was centrifuged at 8,000 x g for 5 min after induction. The pellet was resuspended in 10 ml PBS and placed in an ice bath for ultrasonic lysis (200 W, 5 sec, 5 sec). The lysate was centrifuged at 10,000 x g for 5 min and supernatant was collected for further work.
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To simulate the real environment of our insole, we used the raw ultrasonic cell lysate for bacterial inhibition test. Staphylococcus aureus were grown in LB at 37℃. A dilution of Staphylococcus aureus (20µl; OD600=0.5) was taken and added to 1.5ml, 1.0ml and 0.5ml ultrasonic lysate containing recombinant CecropinXJ respectively and then 0.5ml, 1.0ml and 1.5ml of fresh LB was added accordingly. (Table 1) After incubation at 37℃ for 0.5h, 1.0h and 1.5h, the absorbance of culture at 600nm (OD600) was detected respectively using spectrophotometer (Fig.4).
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Table 1 Different conditions of bacterial inhibition test
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[[File:SCU-China 12.png|450px|thumb|left|“XJa” and “XJb” were the ultrasonic lysate of two repeated independent induced experiments. “Wild” was the ultrasonic lysate of wild BL21(pLysS) strain. Detailed component of each tube was listed. Chloromycetin (C+) was used for positive control (PC) . The ultrasonic lysate of wild BL21(pLyss) was used for negative control (NC). Each tube was cultured at 37℃ with rotation in a speed of 300rpm.]]
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[[File:SCU-China 13.png|450px|thumb|left|'''Fig.4''' The Antimicrobial Test Results. “XJa” and “XJb” were the ultrasonic lysate of two repeated independent induced experiments. “Wild” was the ultrasonic lysate of wild BL21（pLyss). “1.5” and “0.5” means the adding amount of corresponding ultrasonic lysate. Detailed components in each tube were listed in table 1. Chloromycetin (C+) was used for positive control (PC). The ultrasonic lysate of wild BL21(pLysS) was used for negative control (NC). Each tube was cultured at 37℃ with rotation in a speed of 300rpm. The measuring error is within ±0.02.]]
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For the inhibition zone assay, we used the filter paper method. The filter paper was soaked in the raw ultrasonic lysate for 10min and put on the medium after plate coating of Staphylococcus aureus. The antibacterial efficacy and inhibition zone was shown in Fig.5.
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[[File:SCU-China 14.png|450px|thumb|left|'''Fig.5''' Antimicrobial activity of recombinant CecropinXJ using inhibition zone assays. Staphylococcus aureus were plated on LB medium. (A)①25μg/ml chloromycetin was used as positive control. ②250μg/ml chloromycetin was used as positive control. ③10μg/ml kanamycin was used as positive control. ④Ultrasonic lysate of wild BL21(pLyss) was used as negative control. ⑤,⑥Ultrasonic lysates from two repeated independent induced experiments. (B)①Ultrasonic lysates of pET32-CecropinXJ BL21(pLyss). ②Ultrasonic lysate of wild BL21(pLyss) was used as negative control. ③250μg/ml Chloromycetin was used as positive control. ④25μg/ml chloromycetin was used as positive control.]]
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Compared with the high bacterial inhibition effects of prokaryotic expressed CecropinXJ, shown in previous researches, [8] our results show that the recombinant CecropinXJ has limited antimicrobial activity. Even we used three specialized E.coli BL21 strain (RIPL, Rosetta and pGr07) provided by SCUT-China_A, the results are still not ideal. We hypothesized that the induction condition used in our experiments were slightly different with the original research, which lead to serious consequence. However, due to time limits, our experiments on the prokaryotic expression of CecropinXJ had to be paused here. Further experiments using different induction condition are required. Earlier studies had indicated that CecropinXJ shares a similar structure with ABP-CM4, which has the ability to form specific amphipathic α-helices which allows targeting of nonpolar lipid cell membranes. Upon membrane targeting, the helices form ion-permeable channels, subsequently resulting in cell depolarization, irreversible cytolysis and cell death [11-13].
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[10] Cabral KM, Almeida MS, Valente AP, Almeida FC and Kurtenbach E: Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inef ciency of the STE13 protease. Protein Expres Purif 31: 115-122, 2003.
[10] Cabral KM, Almeida MS, Valente AP, Almeida FC and Kurtenbach E: Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inef ciency of the STE13 protease. Protein Expres Purif 31: 115-122, 2003.
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[11] Pokorny A, Kilelee EM, Wu D and Almeida PF: The activity of the amphipathic peptide delta-lysin correlates with phospholipid acyl chain structure and bilayer elastic properties. Biophys J 95: 4748-4755, 2008.
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[12] Wimley WC: Describing the mechanism of antimicrobial peptide action with the interfacial activity model. ACS Chem Biol 5: 905-917, 2010.
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[13] Rausch JM, Marks JR, Rathinakumar R and Wimley WC: Beta-sheet pore-forming peptides selected from a rational combinatorial library: mechanism of pore formation in lipid vesicles and activity in biological membranes. Biochemistry 46: 12124-12139, 2007.