Difference between revisions of "Part:BBa K3420001"

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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

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.

[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