Difference between revisions of "Part:BBa K1166003"

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His-TAT

A biobrick that includes the TAT signal peptide that can be fused to any protein for an easy internalization into mammalian cells (Vivès, et al., 1997). It includes a 6x His-tag for easy purification. TAT is a cell penetrating peptide (CPP) derived from the transactivator of transcription from HIV and has been used to internalize a range of large and small molecules from peptides, DNA and antibodies, to liposomes.

Results: Internalization

Protein expression and confirmation

All of the proteins produced in the internalization module (HIS-GFP and HIS-TAT-GFP) were HIS6x tagged, so we purified them using HisPur Ni-NTA Purification Kit (Thermo Scientific), followed by a Western Blot analysis using an anti-HIS6x antibody HRP conjugated.

Figure 1 shows the assay where we tested the expression of internalization proteins in both the soluble and the insoluble fraction of E.coli BL21 lysates; this way, we could detect which proteins are being correctly expressed.

Figure 1: Western Blot, probed with anti-His6x antibody HRP conjugated. Protein samples were recovered from either soluble or insoluble fractions of E.coli BL21 lysates, and purified using HisPur Ni-NTA Purification Kit (Thermo Scientific). Lane2: Negative control (non-transformed E.coli BL21); Lane3: Positive control (previously confirmed HIS-GFP); Lane4: 0.1 µg of purified HIS-GFP; Lane5: 0.5 µg of purified HIS-GFP; Lane6: 1 µg of purified HIS-GFP; Lane7: 5 µg of purified HIS-GFP; Lane8: Purified HIS-TAT-GFP; Lane9: Amersham High-Range Molecular Weight Marker; Lane10: Non-purified HIS-TAT-GFP

Internalization in vivo assay

The protein samples previously confirmed by Western blot analysis were sterilized by filtration and the Internalization assay protocol was followed as indicated in the Internalization methods. Then, the mammalian cell (NIH and Caco-2) lysates were analyzed by Western blot using an anti-HIS6x antibody HRP conjugated.

In Figure 1 we show the Western blot from the NIH cell lysates at different concentration (1, 5 and 10µg/mL) of purified HIS-GFP and HIS-TAT-GFP. The assay reveals some unclear bands at the expected molecular weight, however, these results are not conclusive and need further testing.

Figure 2: Western Blot, probed with anti-His6x antibody HRP conjugated. NIH cell lysates treated with varying concentrations of purified HIS-GFP and HIS-TAT-GFP expressed in E.coli BL21. Lane1: Positive control (previously confirmed HIS-GFP); Lane2: 10µg/mL treatment with HIS-TAT-GFP; Lane3: 5µg/mL treatment with HIS-TAT-GFP; Lane4: 1µg/mL treatment with HIS-TAT-GFP; Lane5: 10µg/mL treatment with HIS-GFP; Lane6: 5µg/mL treatment with HIS-GFP; Lane7: 1µg/mL treatment with HIS-GFP; Lane8: No treatment; Lane9: Amersham High-Range Molecular Weight Marker

Figure 2 shows the Western blot of the Caco-2 lysates at the same concentrations of the NIH experiment of HIS-GFP and HIS-TAT-GFP. This assay reveals that no internalization proteins were detected in the Caco-2 lysates.

Figure 3: Western Blot, probed with anti-His6x antibody HRP conjugated. Caco-2 cell lysates treated with varying concentrations of purified HIS-GFP and HIS-TAT-GFP expressed in E.coli BL21. Lane1: Positive control (previously confirmed HIS-GFP); Lane2: 10µg/mL treatment with HIS-TAT-GFP; Lane3: 5µg/mL treatment with HIS-TAT-GFP; Lane4: 1µg/mL treatment with HIS-TAT-GFP; Lane5: 10µg/mL treatment with HIS-GFP; Lane6: 5µg/mL treatment with HIS-GFP; Lane7: 1µg/mL treatment with HIS-GFP; Lane8: No treatment; Lane9: Amersham High-Range Molecular Weight Marker

The only evident bands from both Western blots were those from the positive controls, indicating that the western blot protocol was realized correctly. In conclusion, we consider that the lysis of the mammalian cells was very aggressive and it damaged a fraction of the proteins inside the cell. This can be confirmed by the analysis of the concentration of protein of the lysates by Biuret method (Data not show, all the absorbances were under a blank lecture).

References

Vivès E, Brodin P, Lebleu B. (1997). A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem. 272(25):16010-7

Sequence and Features