Part:BBa_K3420005
T7-ferritin-inka-Pak4cat-SV40 (poly A)
T7 promoter is a very specific promoter that is transcribed only by specific T7 RNA polymerase. The ferritin protein has been conjugated with iron, it functions as a novel hyperthermia material. The inka-Pak4cat would allow a higher conjugation of ferritin protein. Here we choose to use SV40 (poly-A) terminator as a DNA sequence with transcription termination effect.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1527
Illegal SapI site found at 949
1. Ft-PAK4 protein crystals grow inside mammalian cells
The Previous study has identified Inka1 as a potent inhibitor of PAK4, which contains two copies of the kinase inhibitory domain and these small regions of themselves can support PAK4cat crystal formation in cells [2]. When inka-box complexes with PAK4cat, conformational changes cause the complex to spontaneously crystallize, producing long rod-shaped crystals[1]
We generated the iron storing construct driven by promoter T7 (BBa_I712074) by fusing ferritin to the N-terminal inkabox portion of the inkabox-PAK4cat(BBa_K3420001) plasmid, which we call Ft-PAK4(BBa_K3420003). HEK293 cells, human embryonic kidney cells 293, a specific cell line originally derived from human embryonic kidney cells grown in tissue culture, are commonly used in cell biology research, because of their reliable growth and propensity for transfection. HEK293T cells derived from the HEK293 cell line expressing a mutant version of the SV40(BBa_K2796012) large T antigen. Due to they can continue to express the SV40 antigen, the cells are often used in transfection experiments with high transfection efficiency. According to Baskaran et al., HEK293T cells transfected with the control inka-PAK4 plasmid grow with a single needle-like crystal. In our experiment, we can observe that Ft-PAK4 protein expression usually begins in 24 hours after transfection, but this only happens if a small amount of the control inka-PAK4 plasmid co-transfected, and their growth lasts around 48 to 72 hours.
To make the crystals more easily visible, we also structured the Wt-PAK4 construct, which replaces ferritin with green fluorescence protein (GFP)( BBa_K3183011). With the same results, the first crystals appeared within 24 hours after transfection and continued to grow up to 72 hours after transfection. We observed that intracellular crystals were morphologically different, that is, the Ft-PAK4 crystals grew slower and shorter but thicker than Wt-PAK4 crystals.
We harvested all crystals in 72 hours after transfection. To characterize isolated crystals, we used a lysis buffer which can break the cell membrane and crystals were subsequently released. And then, the crystals were separated by slow centrifugation, allowing most of the crystals to precipitate. After the supernatant discarded, the crystal-containing pellet was resuspended in buffer containing 0.1 M HEPES.
We checked crystal yield by pipetting a few microliters of suspension onto a coverslip and observing under the microscope. Qualitatively, the Ft-PAK4 crystals tended to be a longer and thicker structure than the Wt-PAK4 crystals. It seemed a little bit different from the situation when expressed in cells. It may be caused by the ferritin-in-core structure. These results imply that ferritins enclosed by PAK4 improve the stubbornness of the crystals, while the Wt-PAK4 crystals are much more fragile when being scraped, centrifuged and suspended.
To test whether the ferritin subunit in the isolated PAK4 crystals could store iron, we carried out the following experiment. Ferritins can catalyze the oxidation of Fe2+ to Fe3+ [3]. Based on it, we added a fresh FeSO4 solution to isolated crystals to wash out free Fe2+, and the final suspension changed color to pale yellow. To verify iron loading, the crystals were assayed with Prussian blue (PB) staining for the presence of mineralized Fe3+. The highly colored PB can prove the presence of iron.
As shown in figure 3.5, the Ft-PAK4 crystals turned a deep blue color in the center while the Wt-PAK4 crystals without ferritin appeared slightly blue on the edges. This result confirms that ferritins are necessary for the Ft-PAK4 crystals to load iron.
Magnetic force microscopy (MFM) measurements were performed by a Bruker Dimension Icon (Santa Barbara, CA, USA). Before the MFM experiment, a normal atomic force microscopy (AFM) experiment in tapping mode was carried out on the samples to reveal the accurate surface topography. The probe used for imaging the surface topography was a non-magnetic probe (Olympus). After the topographic measurement, lifted the cantilever up at a certain distance above the surface of the sample in the second pass to measure the magnetic force between the probe and the sample. Magnetic measurement was conducted using a magnetic MESP probe (Bruker) with the tip radius of ~ 35 nm, the normal spring constant of ~ 2.8 N/m, and the resonance frequency of ~ 75 kHz. Magnetic data was recorded by changes in the amplitude and phase of the probe oscillation.
The Ft-PAK4 crystals, after loading irons, indeed has magnetic response signals according to the results shown in Figure 3.5. The magnetic response signal strength increases as the tip-to-sample distance decreases. The results show that these protein crystals can be attracted by the applied magnetic field and move towards the magnet.
We have shown that the Ft-PAK4 protein could assemble for magnetic sensing and generate substantial magnetic forces. This protein is a genetically encoded ferritin-containing protein crystal that grows inside mammalian cells. We postulate that this well-designed Magnetic Protein Crystal (MPC) could be modified into a more targeted tool. By applying these internal proteins into magnetic hyperthermia therapy, the heating doses can be deposited using minimal control and be selectively localized within the tumor area.
Based on the current research progress, the Ft-PAK4 system cannot realize the magnetic control of genetic targeting in vivo. The following work is to solve iron-load problem through other targeted carriers, and regulate the expression of Ft-PAK4 by optimizing the specific expression promoter.
Protocol
1.HEK293T Cell Culture and Transfection
HEK293T cells are maintained in high-glucose DEME (Dulbecco’s Modified Eagle Medium, Thermo Fisher Scientific) with 1% P/S (Penicillin-Streptomycin, Thermo Fisher Scientific) and 10% FBS (fetal bovine serum, Thermo Fisher Scientific). Before seeding, the cells are cultured on flask. Then, cells are plated at 50,000 cells per well in a 6-well plate. After overnight incubation, cells are transfected by using Lipofectamine 2000 (Thermo Fisher Scientific).
2.Protein Crystals Purification
Pipetted out medium from wells, and rinsed once with sterile water. Added lysis buffer (1% NP-40 in 0.1M HEPES at pH 7.4) directly to the cell culture well and scraped cells off the surfaces. Pipetted the resulting cells into labeled eppendorf tubes and placed on rocker (KS250 basic, Ika labortechnik Staufen) at room temperature for 40 minutes. After rocking, centrifuged at 2000 rpm for 1 minute on a benchtop centrifuge (Micro Star 12, VWR). Discarded supernatant and resuspended pellet in 0.1M HEPES buffer. Check crystal yield by pipetting a few microliters of suspension onto a coverslip and observing under the microscope.
3.Iron Loading
For iron loading, added fresh prepared FeSO4 solution (50 mM, 100X) to a 100 μL aliquot of the crystal suspension, incubated at room temperature for 2 hours. The final suspension changed color to pale yellow. To verify iron loading, freshly prepared the Prussian Blue (PB) stain (Sigma-Aldrich), pipetted a 10 µL droplet of the iron-loaded crystals onto a glass slide or coverslip and added 1 µL of the PB stain. The droplet can be immediately checked by brightfield optical microscopy.
4.MFM
Magnetic force microscopy (MFM) measurements were performed by a Bruker Dimension Icon (Santa Barbara, CA, USA). Before the MFM experiment, a normal atomic force microscopy (AFM) experiment in tapping mode was carried out on the samples to reveal the accurate surface topography. The probe used for imaging the surface topography was a non-magnetic
Reference
[1]Thomas L. Li, Zegao Wang, He You, Qunxiang Ong, Vamsi J. Varanasi, Mingdong Dong, Bai Lu, Sergiu P. Paşca, and Bianxiao Cui Nano Letters 2019 19 (10), 6955-6963
[2] Baskaran, Y.; Ang, K. C.; Anekal, P. V.; Chan, W. L.; Grimes, J.M.; Manser, E.; Robinson, R. C. An in Cellulo-Derived Structure of PAK4 in Complex with Its Inhibitor Inka1. Nat. Commun. 2015, 6, 8681.
[3] Honarmand Ebrahimi, K.; Hagedoorn, P.-L.; Hagen, W. R. Unity in the Biochemistry of the Iron-Storage Proteins Ferritin and Bacterioferritin. Chem. Rev. 2015, 115 (1), 295−326.
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