Bacterial Virus Lambda Gpd-Fusions to Cathelicidins, α- and β-Defensins, and Disease-Specific Epitopes Evaluated for Antimicrobial Toxicity and Ability to Support Phage Display
Abstract
1. Introduction
2. Materials and Methods
2.1. Strains
2.2. Growth Medium and General Dilution “Buffer”
2.3. Plasmid Construction; D-Fusion Plasmids and Encoded Amino Acids
2.4. Single-Burst LDP-Vaccine and SEV Production
2.5. Infection-Complementation Using Transformed Cells
2.6. Complementation Methodology
2.7. Complementation Versus Reversion, Versus Marker rescue
2.7.1. D-Amber Reversion
2.7.2. D-Amber Marker Rescue
2.7.3. Complementation Temperature
2.8. Protein Gels and Western Blots
3. Results and Discussion
3.1. Temperature Dependent Gene Expression from Plasmid pcIpR-GOI-Timm
3.2. Evaluating D-Fusion Cathelicidins for Cellular Toxicity
3.3. Temperature-Dependent Complementation for Phage Growth in D-Defective Phage mutant by Plasmids Expressing gpD or gpD-Fusions
3.4. Evaluating the Influence of D-fusion Size and Toxicity on LDP Production
4. Further Considerations and Summary
Supplementary Materials
Acknowledgments
Conflicts of Interest
Abbreviations
References
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D-Fusion Name (Plasmid Number) | |
---|---|
COOH-terminal additions: a | NH2-terminal additions: a |
His-TAGZ-Dcoe-TEV-LL37 (p619) b | LL37-TEV-Dcoe-His (p620) |
Dcoe-LL37 (p627) c | LL37-Dcoe (p617) c |
Dcoe-PR39 (p625) c | PR39-Dcoe (p623) c |
Dcoe-DEFB126ΔC (p618) d | DEFB126ΔC-Dcoe (p622) d |
Dcoe-HBD3 (p616) | HBD3-Dcoe (p624) |
Dcoe-HD5 (p628) | HD5-Dcoe (p621) |
Dcoe-HNP1 (p615) [see text] | HNP1-Dcoe (p626) [see text] |
Dcoe-YML (p674) | YML-Dcoe (p676) |
D-YML (p675) | His-Dcoe (p614) |
Dcoe-5EV2 (p521,p629) e | His-Dcoe- (p614*) f |
Plasmid In E. coli, Encoded D or D-Fusion Protein | Non-Induces (0 Time) | Non-Induced, 140 min Cell Growth at 30 °C | Induced, 140 min Cell Growth after Shifting Culture to 42 °C |
---|---|---|---|
p613, gpDcoe | 0.10 | 0.44 | 0.79 |
p627, gpD-PR39 | 0.14 | 0.51 | 0.70 |
p625, gpD-LL37 | 0.15 | 0.71 | 0.70 |
Strain 594[pcIpR-D-fusion-timm] Plasmid Construct a | Relative Culture Viability at 42 °C (D-fusion Toxicity) b | EOP c of λimm434 Dam123 at 42 °C on Assay Strain d |
---|---|---|
p613: Dcoe | 0.65 | 0.56 |
p613*: Dcoe- (67 bp deletion within D) e | 0.79 | <0.0001 |
p614: His-Dcoe (11AA N-terminal) | 0.09 | <0.0001 f |
p614*: His- Dcoe-:*(Pro48Glu mutation in D) | 0.87 | <0.0001 |
p615: Dcoe-HNP1 (35AA C-term.) | 0.83 | <0.0001 f |
p626: HNP1- Dcoe (36AA N-term.) | 1.00 | <0.0001 f |
p616: Dcoe-HBD3 (50AA C-term.) | <0.0001 | <0.0005 |
p624: HBD3- Dcoe (53AA N-term.) | 0.78 | <0.0001 f |
p627: Dcoe-LL37 (45AA C-term.) | 0.0002 | g |
p617: LL37-Dcoe (46AA N-term.) | 0.93 | <0.0001 f |
p618: Dcoe-DEFB126-Δ (84AA C-term.) | 0.0001 | g |
p622: DEFB126-ΔC- Dcoe (85AA N-term.) | 0.85 | <0.0001 f |
p619: His-TAGZ-Dcoe-TEV-LL37 (11AA N-, 55AA C-term.) | 0.0003 | <0.000 1f |
p620: LL37-TEV- Dcoe-His (50AA N-, 12AA C-term.) | 0.87 | <0.0001 f |
p628: Dcoe-HD5 (40AA C-term.) | 0.0034 | <0.0001 f |
p621: HD5- Dcoe (41AA N-term.) | 0.77 | <0.0001 f |
p625: Dcoe-PR39 (48AA C-term.) | <0.0001 | g |
p623: PR39-Dcoe (49AA N-term.) | 0.97 | <0.0001 f |
p674: Dcoe-YML (17AA C-term.) | 0.05 | ≥0.90 h |
p675: D(wild type)-YML (17AA C-term.) | 0.88 | 0.93 |
p676: YML-Dcoe (15AA N-term.) | 1.00 | 0.06 |
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Hayes, S. Bacterial Virus Lambda Gpd-Fusions to Cathelicidins, α- and β-Defensins, and Disease-Specific Epitopes Evaluated for Antimicrobial Toxicity and Ability to Support Phage Display. Viruses 2019, 11, 869. https://doi.org/10.3390/v11090869
Hayes S. Bacterial Virus Lambda Gpd-Fusions to Cathelicidins, α- and β-Defensins, and Disease-Specific Epitopes Evaluated for Antimicrobial Toxicity and Ability to Support Phage Display. Viruses. 2019; 11(9):869. https://doi.org/10.3390/v11090869
Chicago/Turabian StyleHayes, Sidney. 2019. "Bacterial Virus Lambda Gpd-Fusions to Cathelicidins, α- and β-Defensins, and Disease-Specific Epitopes Evaluated for Antimicrobial Toxicity and Ability to Support Phage Display" Viruses 11, no. 9: 869. https://doi.org/10.3390/v11090869
APA StyleHayes, S. (2019). Bacterial Virus Lambda Gpd-Fusions to Cathelicidins, α- and β-Defensins, and Disease-Specific Epitopes Evaluated for Antimicrobial Toxicity and Ability to Support Phage Display. Viruses, 11(9), 869. https://doi.org/10.3390/v11090869