Part:BBa_K3420001
iBox-Pak4cat
PAK4 is a kinase with strong links to cellular transformation. 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 [1]. When inka-box(iBox) complexes with PAK4cat, conformational changes cause the complex to spontaneously crystallize, producing long rod-shaped crystals[2].
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 950
Illegal SapI site found at 372
1.Ferritin-iBox-PAK4
Ferritin(BBa_K3420000) is a universal intracellular protein that stores iron and releases it in a controlled fashion. Ferritin is found in most tissues as a cytosolic protein, but small amounts are secreted into the serum where it functions as an iron carrier. We engineered ferritin into the hollow channel in inka-PAK4 crystal by expressing them together. Its structure is illustrated in Figure 1.With ferritin, the crystal is capable of mineralizing substantial amount of iron.
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. 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.
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.
Reference
[1] 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.
[2]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
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