Difference between revisions of "Part:BBa K1166004"

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sTRAIL

Contains the T7 expression cassette for a soluble part of TRAIL with an N-terminal histidine tag (x6) to enhance its purification. It is known to cause apoptosis in various cancer cell lines with minimal cytotoxicity toward normal cells (Walczak, et al., 1999).

TRAIL (TNF Related Apoptosis Inducing Ligand) induces apoptosis by binding to two death receptor domains, TRAIL-R1(DR4) and TRAIL-R2(DR5) (Schneider, et al., 1997). The binding of TRAIL triggers the trimerization of the death receptors that recruit and activate FADD a death domain-containing protein, FADD then recruits and activates caspase-8, leading to the formation of the death-inducing signaling complex (DISC) (Zhang L, et al., 2005).

Results: Therapeutic proteins

Expression of therapeutic proteins

The presence of the therapeutic proteins TRAIL and TAT-APOPTIN in cell lysates was confirmed by Western Blot analysis. Protein samples were obtained from 2 different batches of Escherichia coli BL21 (DE3) cultures, from both the soluble and insoluble products of the sonicated culture.

Figure 1: Western Blot, probed with anti-His6x antibody HRP conjugated. Protein samples were recovered from soluble fractions of E.coli BL21 (DE3) lysates, unless otherwise stated. Lane2: Negative control (non-transformed E.coli BL21); Lane3: Positive control (previously confirmed HIS-GFP); Lane4: HIS-TAT-APOPTIN (from Batch 2, transformant 2); Lane5: HIS-TAT-APOPTIN (from Batch 2, transformant 1); Lane6: HIS-TRAIL (from Batch 2, insoluble fraction); Lane7: HIS-TRAIL (from Batch2); Lane8: Amersham High-Range Molecular Weight Marker; Lane9: HIS-TAT-APOPTIN (from Batch 1); Lane10: HIS-TRAIL (from Batch 1, insoluble fraction)

In Figure 1, we show the expression of TRAIL and TAT-APOPTIN from different cultures of E.coli BL21.

References

Schneider P, Thome M, Burns K, Bodmer JL, Hofmann K, Kataoka T, Holler N, Tschopp J. (1997). TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-kappaB. Immunity. 7(6):831-6.

Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH. (1999). Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med. 5(2):157-63.

Zhang L, Fang B. (2005). Mechanisms of resistance to TRAIL-induced apoptosis in cancer. Cancer Gene Ther. 12(3):228-37.

Sequence and Features