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REFOLD| Article | |
|---|---|
| Title | TRAIL-CM4 fusion protein shows in vitro antibacterial activity and a stronger antitumor activity than solo TRAIL protein. |
| Abstract | A TRAIL-CM4 fusion protein in soluble form with tumor selective apoptosis and antibacterial functions was expressed in the Escherichia coli expression system and isolated through dialysis refolding and histidine-tag Nickel-affinity purification. Fresh Jurkat cells were treated with the TRAIL-CM4 fusion protein. Trypan blue staining and MTT analyses showed that, similar to a TRAIL positive control, Jurkat cell proliferation was significantly inhibited. Flow cytometry analyses using Annexin V-fluorescein revealed that Jurkat cells treated with the TRAIL-CM4 fusion protein exhibited increased apoptosis. Laser confocal microscopy showed that APB-CM4 and the fusion protein TRAIL-CM4 can bind to Jurkat cell membranes and initiate their destruction. ABP-CM4 enhances the antitumor activity of TRAIL by targeting and damaging the tumor cell membrane. In antibacterial experiments, agar well diffusion and bacterial growth inhibition curve assays revealed concentration-dependent TRAIL-CM4 antibacterial activity against Escherichia coli K12D31. The expressed TRAIL-CM4 fusion protein exhibited enhanced antitumor and antibacterial activities. Fusion protein expression allowed the two different proteins to function in combination. |
| PubMed ID | 26926590 |
| Author | Sang Ming, Zhang Jiaxin, Li Bin, Chen Yuqing |
| Journal | Protein expression and purification. 122 82-9 |
| Date | 2016 Apr |
| Protein | |
|---|---|
| Protein name | TRAIL |
| Amino acid sequence | TSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSW ESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYT SYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASF FGAFLVG |
| Comment | His-tag-TRAIL(95-281)-(G3S)3-CM4(1-35) |
| UniProt ID | P50591 |
| Function | Cytokine that binds to TNFRSF10A/TRAILR1, TNFRSF10B/TRAILR2, TNFRSF10C/TRAILR3, TNFRSF10D/TRAILR4 and possibly also to TNFRSF11B/OPG. Induces apoptosis. |
| Domain | Extracellular domain |
| Protein | |
|---|---|
| Protein name | CM4 |
| Amino acid sequence | RWKIFKKIEKVGQNIRDGIVKAGPAVAVVGQAATI |
| Comment | His-tag-TRAIL(95-281)-(G3S)3-CM4(1-35) |
| UniProt ID | P14666 |
| Function | Cecropins have lytic and antibacterial activity against several Gram-positive and Gram-negative bacteria. |
| Domain | Full length |
| Experiment | |||
|---|---|---|---|
| Refolding method | ☐ dilution | ☐ column:filtration | ☐ high pressure |
| ☑ dialysis | ☐ column:binding | ☐ other method | |
| pH | 8.0 | ||
| Temperature℃ | 4 | ||
| Validation | ☑ activity | ☐ solubility | ☑ non aggregability |
| ☐ circular dichroism | ☐ fluorescence tryptophan | ☐ nuclear magnetic resonance | |
| ☐ crystallization | ☐ structure determination | ||
Copyright © 2016 National Institute of Genetics
