Part:BBa_K3351014

HisTag-PhaP-linker-HD5-HisTag

Summary

PhaP contains a hydrophobic granule binding domain and a cytosol-facing hydrophilic domain. PhaP-tagged proteins could interact with various types of hydrophobic surfaces. A (Gly4Ser2)2 flexible linker is inserted between PhaP and HD5 to facilitate attaching of HD5 on surface and displaying sufficient flexibility of this peptide.

Research:

PhaP contains a hydrophobic granule binding domain and a cytosol-facing hydrophilic domain. Complete biodegradable nature of PhaP. PhaP-tagged proteins could interact with various types of hydrophobic surfaces. A (Gly4Ser2)2 flexible linker is inserted between PhaP and HD5 to facilitate attaching of HD5 on surface and displaying sufficient flexibility of this peptide.
A human antimicrobial peptide with 32 amino acids.HD5 fights against Gram-negative bacteria by disrupting and damaging the membrane and penetrating into the cytoplasm. Different oligomeric states were found to perform different degrees of antimicrobial activity. In solution, dimeric and tetrameric states are generally observed. A dimer was found to be the most active form, whereas a tetramer is the largest stable found in solution.Moreover, HD5 also shows anti-endotoxin properties by binding to bacterial lipopolysaccharide (LPS) resulting in LPS neutralization. Both dimer and tetramer seem to induce the LPS membrane disruption.

Figure.1

Imagine:

The fusion protein PhaP-HD5 was expressed in E. coli strain Transetta (DE3).Because PhaP-tagged proteins could interact with various types of hydrophobic surfaces. PhaP-HD5 will anchor to hydrophobic polymer surface efficiently to make band-aids with antibacterial functions.

Design/build:

1.HD5: Mining Gene Information from the Literature.
2.The HD5 fragment was subcloned into the pWDX plasmid using the HindⅢ and SpeⅠ sites to construct the pWDX-HD5 plasmid.

Figure.2

3.The plasmids were then transformed into E. coli BL21(DE3), and transformant clones were screened.
4.E. coli BL21 (DE3) was used as the protein expression host.
5.Extraction and Purification of Proteins (PhaP-HD5) from E. coli BL21 (DE3).
6.The in vitro antibacterial activities of PhaP-HD5 was measured against S. aureus using the filter paper disc diffusion method.
7.PhaP-HD5 will be adhered to the surface of PHA.
8.The bactericidal activity of PhaP, PhaP-HD5, and their coated PHA films will be tested against selected bacteria.

Research

1.Positive colonies (E. coli BL21 (DE3) pWDX(+)::PhaP-HD5) from September 12, 2020 were transferred to test tubes with 2 mL LB+amp and incubated at 37°C, 200 rpm overnight. One 250 mL Erlenmeyer flask with 100 mL LB and 100µL amp was inoculated with 1 mL of one culture above in a 250 mL flask, which both were incubated at 37°C, 200 rpm. OD measurement after three hours showed an OD(600) = 0.6. E. coli BL21 cultures were induced with 100 µL IPTG for incubated at 37°C, 200 rpm overnight. The bacterial growth curves at OD600 were recorded at 30-min intervals for 15 hours.

Figure.3 growth curve

Figure.4

2.The incubated medium was transferred to 50 mL centrifuge tube and the supernatant collected after centrifuging (6791×g) for 3 min at 4°C. After removal of the supernatant by centrifugation (6791×g, 15 min), it was collected precipitate.
3.The bacteria were crushed with an ultrasonic (ultrasonic power: 400 W, crushing time: 1 s, interval time: 1s) on ice for 2 min. (A volume of 20 ml of the Bacterial lysis solution was added to each baterial precipitat, Bacterial lysis solution 2.0: 20mM/L tris-HCl 150mM/L NaCl)
4.PhaP-HD5 was not observed in the supernatant analyzed by SDS-PAGE.

Research and design cycle:

Through analysis, the reason we thought was the Bacterial lysis solution or the ultrasonic power/total time,so we change the Bacterial lysis solution and ultrasonic total time.
1.The bacterias were crushed with an ultrasonic (ultrasonic power: 400 W, crushing time: 1 s, interval time: 1s) on ice for 15 min. (A volume of 20 ml of the Bacterial lysis solution was added to each baterial precipitat, Bacterial lysis solution 2.0: 20mM/L tris-HCl 150mM/L NaCl). But PhaP-HD5 also was not observed in the supernatant analyzed by SDS-PAGE.
2.This time,we increased the power of ultrasound and the time to crush the bacteria,which were crushed with an ultrasonic (ultrasonic power: 500 W, crushing time: 3 s, interval time: 5s) on ice for 30 min.it is pity that we couldn’t observed the PhaP-HD5 in in the supernatant analyzed by SDS-PAGE.
3.So we increased the total time of ultrasound to 45 min, the bacterias were crushed with an ultrasonic (ultrasonic power: 500 W, crushing time: 3 s, interval time: 5s) on ice for 45 min.The sonicated bacterial lysis solution was centrifuged 6791 × g for 15 min at 4°C. Equivalent amounts of supernatant and precipitate were subjected to SDS-PAGE analysis. The supernatant, containing target proteins(PhaP-HD5)

Figure.5

Bacteriostatic experimen

It was noticed that PhaP-HD5 showed the antibacterial effects when examined on S. aureus strain RN4220.

Figure.5

Characterization

Figure.6 growth curve

Reference

[1] Xue Q, Liu XB, Lao YH, Wu LP, Wang D, Zuo ZQ, Chen JY, Hou J, Bei YY, Wu XF, Leong KW, Xiang H, Han J. Anti-infective biomaterials with surface-decorated tachyplesin I. Biomaterials. 2018 Sep;178:351-362. doi: 10.1016/j.biomaterials.2018.05.008. Epub 2018 May 9. PMID: 29778319.
[2] Wang, Cheng et al. “A Simplified Derivative of Human Defensin 5 with Potent and Efficient Activity against Multidrug-Resistant Acinetobacter baumannii.” Antimicrobial agents and chemotherapy vol. 62,2 e01504-17. 25 Jan. 2018, doi:10.1128/AAC.01504-17

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]