We obtained synthetic rat CART 55-102 peptides commercially from different providers (003-62, Phoenix Pharmaceuticals, Burlingame, CA, USA; 46-2-55, American Peptide Company, Sunnyvale, CA, USA; H-4446, Bachem, Bubendorf, Switzerland). Human CART 39-89 peptide and human CART 49-89 peptide were produced recombinantly in Escherichia coli. Briefly, the sequence coding for hCART49-89 and hCART 39-89 was codon-optimized for expression in E. coli, synthesized, and subcloned into the pET32a expression vector (Novagen, Madison, WI, USA). A fusion protein with an N-terminal thioredoxin-A, followed by a HIS-tag and a tobacco etch virus (TEV) protease cleavage site under control of a T7/lac promoter was placed upstream of the CART peptide sequence. We transformed this construct into E. coli BL21(DE3) (Invitrogen, Carlsbad, CA, USA). We used a fed-batch fermentation process in a 7-liter bioreactor (Applikon, Foster City, CA, USA) for expression of the protein. We used minimal salt medium with 60 g/L glycerol. We maintained the temperature at 30°C, pH at 7, and oxygen at 20% saturation. When glycerol was almost depleted in the medium, we added 20g/L lactose for induction of expression. Cells were harvested, centrifuged, and lysed by sonication in a Rosett Cell (Branson, Danbury, CT, USA). Polyethyleneimine (0.2% vol/vol) was added to the lysate and the precipitate was centrifuged. We loaded the supernatant on a Ni-NTA superflow (Qiagen, Hilden, Germany) column, eluted with a buffer containing 500 mM imidazole. The eluate was dialyzed against a 50-mM sodium phosphate buffer (pH 7.3) and the N-terminal tag was cleaved by TEV protease, containing an N-terminal HIStag itself. After purification of the cleaved CART peptide, it was loaded on a Ni-NTA superflow column and the flow-through was concentrated using a VivaSpin concentrator (3000 kDa MWCO; Sartorius Stedim Biotech, Aubagne, France). Endotoxins and other contaminants were removed on a Q sepharose FF column (GE Healthcare, Little Chalfont, UK). Buffer exchange and separation of the different folded CART peptide variants was performed by a size exclusion chromatography (Superdex-30), using 50 mM ammonium acetate. The fractions containing the correctly folded hCART peptide were pooled, checked for endotoxins by a limulus amebocyte lysate (LAL) QCL-1000 assay (Lonza, Basel, Switzerland), and freeze-dried.
We analyzed synthetic and recombinant peptides by nondenaturing polyacrylamide gel electrophoresis (PAGE) in the presence or absence of dithiothreitol (DTT), and liquid chromatography–mass spectrometry (LC-MS) (Supplemental Fig. 1) to determine purity and quality. We performed LC-MS on a XBridge BEH300 C18 column (3.5 µm, 1 × 150mm; Waters) using a gradient of (A) 0.1% HCOOH in MilliQ (Supplemental Fig. 1A) and (B) acetonitrile (Supplemental Fig. 1B) (0–30 minutes, 10% to 30% A; 30–30.1 minutes, 30% to 90% A; 30.1–35 minutes, 90% A; 35–35.1 minutes, 90% to 10% A; 35.1–50 minutes, 10% A). The flow rate was 50 µL/min (MIC splitter). Column temperature was 40°C. We used MicrOTOF-Q (Bruker, Billerica, MA, USA) to determine the mass of the peptides.
We subsequently formulated the peptides in slow-release pellets by Innovative Research of America (IRA, Sarasota, FL, USA). We determined the quality of the peptides in the pellets and the release of the peptides from the pellets by nondenaturing PAGE either in the presence or absence of DTT and LC-MS (Supplemental Fig. 2).