D oligonucleotide encoding nine amino acids from the basic domain of HIV-1 Tat. The sequences were: top strand, 5′-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3′; and bottom strand, 5′-TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC-3′. The doublestranded oligonucleotide was directly ligated into the NdeIXhoI-digested pET15b in frame with the six histidine openreading frames to generate the HisTat expression plasmid, pHisTat. Next, on the basis of the cDNA sequence of human Cu, Zn-SOD, two oligonucleotides were synthesized. The top strand, 5′-CTCGAGGCGACGAAGGCCGTGTGCGTG-3′, contained a XhoI restriction site, and the bottom strand, 5’GGATCCTTATTG-GGCGATCCCAATTAC-3′, contained a BamHI restriction site. The reaction mixture was made up in a 50 siliconized reaction tube and heated at 94 for 5 minutes. The program for PCR consisted of 30 cycles of denaturation at 94 for 40 seconds, annealing at 54 for 1 minute, and elongation at 70 for 3 minutes, and the final extension at 72 for 10 minutes. The PCR products were purified by preparative agarose gel electrophoresis. The purified products were ligated into a TA-cloning vector and then transformed into a competent cell. The bacterial cells (E. coli BL21) transformed with pTat-SOD were harvested and disrupted by sonication in 5 ml binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) containing 6 M urea. After centrifugation, supernatant was immediately loaded onto a 2.5 ml Ni2+nitrilotriacetic acid Sepharose column (Qiagen, Valencia, CA, USA). After the column was washed with 10 volumes of a binding buffer and 6 volumes of a washing buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), the fusion protein was eluted with an elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The fusion protein containing fractions were combined and the salts were removed using PD10 column chromatography. To enhance the transduction efficacy of Tat-SOD, copper ion recovery of overexpressed Tat-SOD was conducted as described previously [16]. We purified six histidine residue-tagged SODs without an HIV-1 Tat PTD by using a SOD expression vector (pSOD) as a control SOD protein. pTat-green fluorescent protein (GFP) was also constructed to express the basic domain of HIV-1 Tat as a fusion with GFP as was described previously [17] and used as a control Tat protein.Materials and methodsReagents Recombinant human IL-1 was obtained from R D systems (Minneapolis, MN, USA). Anti-polyhistidine IgG was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Nitrate/nitrite colorimetric assay kits were purchased from SP600125 price Cayman Chemical (Ann Arbor, MI, USA). Restriction endonucleases and T4 DNA ligase were purchased from Promega (Madison, WI, USA). Pfu polymerase was obtained from Stratagene (La Jolla, CA, USA). TA-cloning vector was obtained from Invitrogen (Carlsbad, CA, USA). Oligonucleotides were synthesized from Gibco BRL custom primers (Carlsbad, CA, USA). Isopropyl–D-thiogalactoside (IPTG) was obtained from Duchefa (Haarlem, the Netherlands). Plasmid pET15b and Escherichia coli strain BL21 (DE3) were from Novagen (Madison, WI, USA). Ni2+-nitrilotriacetic acid Sepharose superflow was purchased from Qiagen (Hilden, Germany). A human PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25962748 Cu, Zn-SOD cDNA fragment was isolatedPage 2 of(page number not for citation purposes)Available online http://arthritis-research.com/content/8/4/RFigureTransduction of transactivator of transcription (Tat)-superoxide dismutase (SOD) fusion protein into monolayer cultured ch.
