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HMG-1 was cloned by DNA amplification of the 648-base pair (bp) open reading frame from Rat Brain Quick-Clone cDNA (5 ng; Clontech, Palo Alto, CA) with the following primers: 5′-CCCGCGGATC-CTCGAGGGAAGGATGGGCAAAGGAGATCCTA-3′ and 5′-CCCGCAAGCTTATTCATCATCATCATCTTCT-3′ (PCR at 94°C for 1 min. 56°C for 2 min. 72°C for 45 s; 30 cycles). The 680-bp PCR product was digested with Bam HI and Hind III and subcloned into the Bam HI-Hind III cloning sites of the pCAL-n vector (Stratagene, La Jolla, CA). The recombinant plasmid was transformed into E. coli BL21(DE3)plysS (Novagen, Madison, WI), and positive clones were confirmed by DNA sequencing of both strands. Transformed cells were induced with isopropyl-D-thiogalactopyranoside, and rHMG-1 protein was purified with a calmodulin-binding resin column (Stratagene). As controls for experiments involving administration of rHMG-1 to mice, we purified proteins from E. coli BL21(DE3)pLysS that had been transformed with a plasmid that lacks the HMG-1 cDNA insert (pCAL-n). The amount of control material administered to mice was normalized to the number of E. coli that produce 0.5 mg of rHMG-1.
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Supplementary data can be found on Science Online at www.sciencemag.org/feature/data/1037699.shl.
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We thank C. Dang for technical assistance; J. Eaton, J. Roth, B. Sherry, M. Bukrinsky, and M. Symons for critical reading of the manuscript; and D. Prieto for administrative assistance.
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