Soluble TRAIL Waspurified Frome. Coli

Supplemental Results

Soluble TRAIL waspurified fromE. coli

To produce a soluble form of TRAIL in E. coli, the C-terminal amino acids 114 – 281 were expressed together with a His-tag and a HA-tag (further on named TRAIL). To achieve highly active TRAIL, an isoleucin zipper motif was added at the N-terminus to support trimer formation (named ILZ-TRAIL) as it has been described previously (1). To produce a dysfunctional control variant, the cysteine at position 230 was replaced by a serine (named TRAIL C230S) in parallel to published data (2) (suppl. figure 1A) as it is known that the cystein 230 is responsible for trimerization and activation of TRAIL (3, 4).

Although TRAIL and ILZ-TRAIL has been purified von E.coli before (5, 6), we found recombinant TRAIL nearly exclusively stored in inclusion bodies, especially large protein variants of TRAIL containing several tags (data not shown). Therefore, TRAIL was isolated from purified inclusion bodies under denaturing conditions in the presence of 8 M urea yielding a high protein recovery from bacteria (up to 10 mg HA-TRAIL was purified from 100 ml culture of bacteria). TRAIL isolated from inclusion bodies achieved purity comparable to Ni-purified protein, rendering additional purification steps unnecessary (suppl. Figure 1B and data not shown). Refolding was performed by dilution as it has been described for other TRAIL variants before(7)and optimal activity was obtained by dialysis to a ZnCl2-containing storage buffer (see Methods for details). This method now allows the quick, easy, efficient and cost-effective production of high amounts of pure and active TRAIL and TRAIL variants.

Refolded soluble TRAIL activated Apoptosis and NF-кB

Recombinant TRAIL induced apoptosis in a dose dependent manner in the TRAIL-sensitive T-cell leukemia cell line JURKAT. To prove that apoptosis induction by the produced TRAIL depended on the specific interaction between TRAIL and its receptors, TRAIL was neutralized by preincubation with a TRAIL R2:Fc fusion protein which completely abrogated TRAIL-induced apoptosis for both ILZ-TRAIL and TRAIL (suppl. Figure 1C and data not shown). Our TRAIL was similarly active compared to commercially available TRAIL (suppl. Figure 1D) and its activity remained stable upon storage for several months (data not shown). In JURKAT cellslow concentrations of ILZ-TRAIL weremore active than low concentrations of TRAIL for induction of apoptosis indicating that forced trimerization increased the apoptotic potential of TRAIL(suppl. Figure1E). To study TRAIL-induced activation of NF-κB, 293T cells were used as they show marked activation of NF-κB by TRAIL with absence of TRAIL-induced apoptosis. TRAIL-induced activation of NF-κB was tested, using a luciferase reporter assay in293T cells. TRAIL induced activation of NF-κB in a dose-dependent manner. Comparable to our data obtained for induction of apoptosis, TRAIL-induced activation of NF-κB was enhanced upon trimerization of TRAIL and abolished, when the physiologic trimerization was inhibited (suppl. Figure 1F).

In contrast, the trimerization-deficient control TRAIL variant (TRAIL C230S) was completely inactive for induction of both apoptosis (suppl. figure 1E) and activation of NF-κB (suppl. Figure 1F) confirming the high importance of trimerization of TRAIL for its activity (2). Control experiments using TRAIL C230S and inhibition of TRAIL by soluble TRAIL-receptor-Fc fusion proteinexcluded unspecific effects induced, e.g., by bacterial contaminations in our preparations(suppl. Figure 1C, E andF).

Taken together, we established an easy, cheap and straightforward method to produce high amounts of TRAIL and TRAIL variants which were functionally active.

Supplemental Material and Methods

TRAIL production

Protein was expressed in E.coli BL21(DE3)9 and induced by use of lactose containing medium for 24 hours and harvested by centrifugation with 4500 rpm for 30 minutes. Bacterial pellet was lysed in BugBuster Master Mix (Merck) and the inclusion bodies were purified according to the manufacturer’s protocol. Inclusion bodies were lysed in Urea buffer (8 M Urea, 100 mM NaH2PO4, 10 mM Tris, pH 8,0) for 1 hour and centrifuged at 10 000 g for 30 minutes at room temperature. Supernatant was stepwise diluted with refolding buffer (100 mM Tris, 700 mM arginine, 500 mM NaCl, 1 mM DTT (7)) to its eightfold volume. Concentration of the solution was performed by ultrafiltration using U-Tubes 20-10 (Merck). The protein solution was finally dialyzed to storage buffer (20 mM Tris-HCl (pH 8,0), 500 mM arginine, 100 mM NaCl, 0,02% Tween 20, 1 mM ZnCl2(8)) using D-Tubes (Merck) with a cut off of 6-8 kDa. Concentration determination was done by comparison with BSA standard in a Coomassie stained SDS gel. For this aimpurified protein was separated in a 12% SDS gel and fixed (34% methanol, 2% phosphoric acid, 17% ammonium sulfate) for 1 hour before staining (0,066% Coomassie Brilliant Blue, 34% methanol, 2% phosphoric acid, 17% ammonium sulfate)for 24hours. LPS content of a TRAIL preparation was <1 ng/µg protein, determined by LAL-Assay at Profos AG (Regensburg, Germany).

Production of lentiviral particles

The third generation packaging plasmids (9) pMDLg/pRRE, pRSV-Rev and pMD2-G were kindly provided by T.Schroeder (Munich, Germany). Stocks of lentiviral particles of vesicular stomatitis virus (VSV) G protein-pseudotyped lentivector were prepared by transient four-plasmid transfection of 293T cells using TurboFect in vitro Transfection Reagent (Fermentas).Virus containing supernatant washarvested after 72 hours, filtrated and concentrated by ultrafiltration.

References

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