The DNA encoding the monomer of the Tn3 in the DRA encoded the following amino acid sequence (GAIEVKDVTDTTALITWAKPWVDPPPLWGCELTYGIKDVPGDRTTIDLQQKHTAYSIGNLKPDTEYEVSLICFDPYGMRSKPAKETFTT) (10). The E. coli codon-optimized gene was purchased as a “G-block” from the Integrated DNA Technologies (IDT). The gene was purchased with a (Gly4Ser)3 linker at the C terminus and designed with restriction sites compatible with recursive directional ligation (RDL) for seamless cloning of oligomeric genes (45). The amplified PCR product was purified using a Qiagen PCR cleanup kit and digested with Bse RI for insertion into a Bse RI/CIP digested pET-24(+) vector modified for RDL. The insert and vector were agarose gel purified and ligated with Quick Ligase to clone the single unit construct. This was followed by digestion of the single unit construct [Tn3 in pET24(+)] with Bse RI/CIP and ligation with Bse RI–digested insert (Tn3 monomer) to clone 2, 4, and 6 Tn3 repeats in the pET-24(+) vector (Novagen, Madison, WI). EB5α cells (EdgeBio) were used for cloning steps. All enzymes were purchased from New England Biolabs.

The gene for the depot-forming ELP (ELPdepot) that encodes the amino acid sequence (VPGVG)120 and the soluble ELP (ELPsoluble) that encodes the amino acid sequence (VPGA/GG)120 was recombinantly fused to the hexameric Tn3 fusions using RDL. Both ELP genes were available from previous studies (35). The DNA sequence of the ELP genes can be obtained from previous publications (27, 35). The RDL method for this particular vector called for digestion of the oligomerized Tn3 in modified pET24(+) with Bse RI and Bgl I and digestion of ELP in pET24(+) with Acu I and Bgl I. The digested fragments of DNA were separated using agarose gel electrophoresis, and the DNA bands of the appropriate molecular weights were excised and gel purified using the QIAquick Gel Extraction kit (Qiagen). The purified fragments were ligated using Quick Ligase (New England Biolabs), and successful clones were identified by DNA sequencing analysis.

The hexamer ELPdepot-DRA fusion constructs were expressed in SHuffle T7 Express cells in 2XYT media in 1-liter shake flasks (New England Biolabs). Overnight cultures (50 ml) were used to inoculate 1-liter Erlenmeyer flasks in a shaker incubator (GYROMAX 747 orbital incubator shaker, Amerex Instruments Inc.), and cells were grown for 4 to 5 hours at 30°C and then induced with 1 mM sterile isopropyl-β-d-thiogalactopyranoside (IPTG) and incubated at 180 rpm at 25°C for another 6 to 12 hours. Cells were pelleted, resuspended in 50 mM tris (pH 8), sonicated, and centrifuged at 15,000 rpm at 4°C for 15 min to separate cell debris from the soluble fraction. Proteins were purified from the soluble fraction of the cell lysate using inverse transition cycling (ITC), a method that exploits the LCST phase transition of ELP fusions and involves repeated cycles of protein aggregation and solubilization (46). Specifically, the “hot spin” of ITC was performed by the addition of <2 M ammonium sulfate until the solution became turbid and the salt was fully dissolved and centrifuged at 14,000 rpm at 35°C for 20 min to pellet the protein. The phase-transitioned protein pellet was then resuspended in 20 mM tris, 300 mM arginine at pH 7 and placed in a rotator at 4°C. The “cold spin” of ITC was performed by centrifuging the protein at 4°C, 14,000 rpm and preserving the supernatant, which contained the ELPdepot-DRA fusion. The hot spin/cold spin process was repeated twice before further purification by size exclusion chromatography on a Superdex HiLoad 26 60/200 column in PBS on an ÄKTA chromatography system (GE Healthcare Life Sciences). Pure eluate then underwent buffer exchange into 20 mM tris, 300 mM arginine (pH 7) using 10-kDa Amicon Ultra centrifugal filters (EMD Millipore). All purified proteins were analyzed by SDS-PAGE on Bio-Rad Mini-PROTEAN TGX Tris-HCl Stain-Free gels for correct molecular weight bands.

For DRA expression without fusion to ELP, the DNA sequence encoding a periplasmic secretion signal, oppA “MTNITKRSLVAAGVLAALMAGNVALA,” was appended at the 5′ terminus of the hexameric DRA gene by the previously discussed RDL method, and DNA encoding a (His)8 tag was appended at the 3′ end of the DRA gene to create a gene that encodes the following construct—oppA-DRA-His8. This construct was then expressed in BL21(DE3) cells: 50 ml of overnight cultures was used to inoculate 1-liter shake flasks, and cells were grown at 37°C for 4 to 5 hours, then induced with 1 mM sterile IPTG, and incubated at 37°C in a shaker incubator (GYROMAX 747 orbital incubator shaker, Amerex Instruments Inc.) for another 4 to 6 hours. Cells were then pelleted and resuspended in 100 ml of ice-cold 10 mM tris, 1 mM EDTA (pH 8) buffer and placed on a rotator at 4°C for 1 to 2 hours to complete protein extraction from periplasmic space. The periplasm extraction samples were then centrifuged at 14,000 rpm for 15 min at 4°C to pellet cell debris. Proteins in solution were precipitated with ammonium sulfate (60%, w/v) and centrifuged for 15 min to pellet the precipitated protein. The protein pellet was then placed on ice and resuspended in ice-cold PBS. Immobilized metal affinity chromatography (IMAC) on a Nickel-NTA agarose resin (Thermo Fisher Scientific) was then used to purify the His-tagged DRA from other periplasmic proteins by following the manufacturer’s protocol. Pure eluate then underwent buffer exchange into 20 mM tris, 300 mM arginine (pH 7) using 10-kDa Amicon Ultra centrifugal filters (EMD Millipore).

TRAIL-His8 (TRAIL amino acids 114 to 281) was purchased as a G-block from IDT, cloned into the pET-24(+) plasmid system (Novagen), and grown in BL21(DE3) cells (EdgeBio). Overnight cultures (50 ml) were used to inoculate 1-liter shake flasks, and cells were grown at 25°C for 4 to 5 h, then induced with 1 mM sterile IPTG, and incubated at 16°C in a shaker incubator (GYROMAX 747 orbital incubator shaker, Amerex Instruments Inc.) overnight. Cells were pelleted, resuspended in 20 mM tris, 100 μM zinc sulfate, 10 mM calcium chloride, 10 mM dithiothreitol (DTT) (pH 7.4), sonicated, and centrifuged at 4°C at 15,000 rpm for 15 min to separate cell debris from the soluble fraction. The His-tagged DRA was purified from other periplasmic proteins protein on a Nickel-NTA agarose resin by IMAC by following the manufacturer’s protocol (Thermo Fisher Scientific). Pure eluate then underwent buffer exchange into 20 mM tris, 100 μM zinc sulfate, 10 mM calcium chloride, and 10 mM DTT (pH 7.4). All chemicals were purchased from Sigma-Aldrich. The EC50 of the in-house TRAIL was within an order of magnitude of commercial TRAIL.

All proteins used for the in vivo studies were endotoxin purified using Pall Mustang E Membrane sterile/endotoxin filters and tested using the GenScript ToxinSensor Single Test Kit endotoxin test to ensure levels below the U.S. Food and Drug Administration–recommended limit of 0.25 EU/ml (47).

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