Part:BBa_K3647222
Contents
pT7-Eosinophil cationic protein
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 109
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
General info
Eosinophil cationic protein (ECP), also known as ribonuclease 3, is a member of the ribonuclease superfamily. Protein is located in the matrix of eosinophil and its release is triggered by an immune stimulus. In humans, ECP is encoded by the RNASE3 gene. ([http://www.sciencedirect.com/science/article/pii/088875439290237M?via%3Dihub#!/ Mastrianni, 1992])
Protein possesses neurotoxic, helmintho-toxic, and has low ribonucleolytic activities. Protein is known to be involved in the antimicrobial humoral response, innate immune response, neutrophil degranulation, RNA catabolic process. ([http://www.researchgate.net/publication/11766282_Eosinophil_Cationic_Protein/ Boix, 2001])
Human eosinophil cationic protein is a single-chain, zinc-containing protein with a molecular weight ranging from 16 to 22 kDa. The ECP amino acid sequence has a 32 % identity to human pancreatic ribonuclease (RNase) A. In general, the RNase activity of ECP is required for its neurotoxic and antiviral properties of the protein, but not for its antibacterial and antihelminthic activities. ([http://www.sciencedirect.com/science/article/pii/B9780323085939000176/ Kita, 2014])
ECP was shown to be correlated correlates well with airway inflammation in asthma during numerous studies. Serum ECP was found to directly correlate with activated eosinophils in bronchial mucosa of adult asthmatic patients. In children serum ECP has also been found to be a more sensitive marker of asthma severity than peripheral blood eosinophil counts in acute exacerbations. ([http://www.sciencedirect.com/science/article/pii/S0954611106004070/ Koh, 2007])
Usage and Biology
Expression system
The part was expressed with a T7-RNA-polymerase promoter (BBa_I719005), as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in Escherichia coli (E. coli) (BBa_K1758106).
Results
Plasmid analysis
Preparation of an agarose gel was done by mixing 1.3 g of agarose in 100 mL of TAE 1X buffer (containing 242 g Tris, 57.1 mL Glacial acetic acid, 100 mL EDTA disodium salt, and filled up to 1L with dH2O). The mixture was microwaved until it boiled and then poured into a mold. Before the gel had hardened, a comb was added to form the wells. To prepare the samples to run on the gel, 7 µL of loading dye was mixed with 7 µL of each DNA sample. EDN, ECP, and HNMT were loaded, as well as 7 µL of a ladder for reference (1 kb plus DNA ladder, New England Biolabs). The gel was run at 100 V for 60 min. After finishing, it was stained with ethidium bromide for 5 min (EtBr solution should be orange, with light shaking). To remove excess EtBr, destaining with TAE buffer (just enough to cover the gel) was done for 1 minute (with light shaking). To analyze the results, the gel was placed in a gel camera (C150, Azure Biosystems). The results show the presence of a plasmid.
Protein expression
Seeds were prepared by spreading 100 µL from a liquid culture of pIDTSmart-ECP E. coli (BL21) onto LB-Miller agar plates with 50 µg/mL kanamycin. A total of 3 plates were made for each construct. These plates were incubated at 37 °C for 16 hours. To inoculate the three plates, 400 µL of LB-Miller was poured onto them and released from the plate using a spreader. Cells from all the plates were then moved into a culture flask with 1 L 2X LB-Miller (20 g peptone, 10 g yeast extract, 10 g NaCl) and 50 µg/mL kanamycin. This inoculation was then left to grow until an OD600 at 0.6 for both of the constructs. The induction of the T7 system was done with 1 mM IPTG and the temperature of the incubator was lowered to 20 °C. The cells were incubated for 16 hours. The optical density (600 nm) at harvest was measured to 5.7 for both of the constructs. Harvest was done by centrifugation at 3500 g 4 °C for 20 minutes using a swing-out rotor. The bacterial pellets were then resuspended using Buffer G (6 M guanidine hydrochloride, 10 mM Tris, 100 mM Na2HPO4, 10 mM reduced glutathione, pH 8.0) and placed at -80 °C.
Bacterial lysis
Bacteria lysis was done using a sonication method. Pellets of harvested cells were resuspended in the appropriate buffer and frozen at -80 °C. The cells were then thawed at 37 °C. A sonication probe was placed in the bacterial slurry and sonication was done at 30 % amplitude with 30 seconds ON 30 seconds OFF for a total ON time of 4 minutes. Samples were kept on ice during sonication to prevent excessive heating of the sample. After sonication samples were centrifuged at 10000 g for 40 minutes. The supernatant was saved for further purification.
IMAC purification
IMAC purification can be used for His-tagged proteins. A volume of 1 mL of Nickel-nitrilotriacetic acid agarose was added to a PD10 column and washed with 10 mL of buffer B (10 mM Tris, 100 mM Na2HPO4, pH 8.0). The lysate was poured onto the IMAC column and flowthrough was saved until protein has been detected in the purified sample. A volume of 10 mL buffer G (6 M guanidine hydrochloride, 10 mM Tris, 100 mM Na2HPO4, 10 mM reduced glutathione, pH 8.0) was added, followed by adding 7 mL of 50 % buffer G and 50 % buffer B, and 7 mL buffer B afterward. Protein was eluted using 10 mL of buffer E (10 mM Tris, 100 mM Na2HPO4, 500 mM Imidazole, pH 5.8). All fractions were then run on an SDS-PAGE gel to verify the purity and presence of the protein.
SDS-PAGE
To prepare samples to run on the SDS-PAGE gel, 20 µL sample was mixed with 18 µL sample buffer (containing 2 mL Tris 1 M, 4.6 mL glycerol, 1.6 mL SDS, 0.8 mL dH20, and bromophenol blue until strong color) and 2 µL β-mercaptoethanol. The mix was heated to 95 °C for 5 minutes. After preparation, the samples were loaded onto the gel. The gel was placed in the gel electrophoresis tray and a 1X running buffer was added (~0.5 L). Voltage was set to 100 V and the gel was run until all samples wandered into the separation gel. Then the voltage was then increased to 200 V and the gel was run until the sample buffer color front reached the bottom. The gel was removed from the gel electrophoresis tray and rinsed with water twice, a final rinse was done with water and heated to near-boiling temperature. Following, the water was removed and the staining solution was added, this solution was heated to a near-boiling temperature as well. The gel was incubated with a staining solution for 30 minutes with light shaking. Afterward, the staining solution was removed and the destaining solution was added and left to destain for 30 minutes with a light shake. The destaining solution was changed once. The results of the SDS-PAGE analysis showed that the protein was successfully expressed. The larger size at 25 kDa (theoretical size, 17.4 kDa) on the gel can be explained by the highly positive protein. NanoDSF was performed afterward to ensure the fold.
Protein dialysis
Protein dialysis was done in order to change the buffer conditions. A membrane (Standard RC tubing, MWCO 3.5 kDa, Spectrum Laboratories) was incubated in dH2O for 5 minutes and samples were loaded into the dialysis tubing. Dialysis was then done in 5 mM EDTA buffer (1 L) for 16 hours at 4 °C. The dialysis was repeated once but using a 5 mM borate buffer (1 L) instead. Some precipitation was seen, but protein concentration in the supernatant was still enough for further analysis.
Nano differential scanning fluorimetry
To investigate the protein fold and stability, nano differential scanning fluorimetry (Prometheus, NanoTemper) was performed. A volume of 10 µL of protein sample was loaded into capillaries (using capillary force) and simultaneously scanned at 330/350 nm wavelengths. The temperature was set to gradient and started at 20 °C, rising 0.5 °C/min until it reached 110 °C. Dithiothreitol was added (10 mM) as a control to find out the state of the 4 disulfide bonds that ECP contains. As seen in Figure 5. the first conformational change occurred around 49 °C (10 mM DTT, 5 mM borate, pH 7.5) and 51 °C (5 mM borate, pH 7.5), which is different from the li.terature value at 39.4 °C. The thermal unfolding of the protein can be seen at 63.9 °C in the control sample with DTT and 68.9 °C in the sample without any DTT. This is in accordance with the literature value at 68.6 °C (50 mM HEPES, pH 7.5)(Nikolovski, 2006). These results indicate that the disulfide bonds and the fold of the protein were at least partially correct. These results also confirm that plasmids were constructed correctly.
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