The cDNA for the mature peptide (amino acid residues 117-268) of rat IL-1β (DDBJ/EMBL/GenBank accession No. NM_031512) was amplified by PCR using the primers 5’-CCAGCATATGGTTCCCATTAGACAGCTGCACTG-3’ and 5’-CCGAATTCAGGAAGACACGGGTTCCATGG-3’. The amplified DNA fragment was digested with Nde I and EcoRI and cloned into the pCold III vector (Takara Bio Inc., Kusatsu, Shiga, Japan). The resultant construct and pLysSRARE2 plasmid (Novagen Inc., Madison, WI, USA) were introduced into Escherichia coli strain BL21 (Takara Bio Inc.). The transformed cells were grown and cold-shocked. The cells were harvested, lysed, and subjected to ammonium sulfate precipitation and dialysis, followed by ultrafiltration to purify the mature peptide, the activity of which was verified by comparison with that of recombinant rat IL-1β (PeproTec, Rocky Hill, NJ, USA).
Pcold 3 vector
Manufactured by Takara Bio
Sourced in Japan
The PCold III vector is a protein expression system designed for the production of recombinant proteins in E. coli. It features a cold-inducible promoter that allows for controlled expression of target proteins at low temperatures, which can improve protein solubility and folding.
Automatically generated - may contain errors
Lab products found in correlation
Escherichia coli strain bl21, by Takara Bio (1 mentions)
Oligonucleotide, by Thermo Fisher Scientific (1 mentions)
Quikchange 2 kit, by Agilent Technologies (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Hitrap imac hp column, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
Xbai, by Takara Bio (1 mentions)
4 protocols using pcold 3 vector
1
Recombinant Rat IL-1β Purification
2
Bacterial Collagen Scl2.28 Protein Engineering
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The DNA sequence of bacterial collagen Scl2.28 was previously codon-optimized and inserted into the pCold-III vector (Takara Bio Inc., Japan) (43 (link)). Oligonucleotides encoding the sequence GARGERGFPGERGVQGPP from the human collagen α1 chain were designed with XmaI and ApaI sticky ends and synthesized (Thermo Fisher Scientific, Waltham, MA). Annealed dsDNA was cloned into pCold vector containing the Scl2.28 sequence that was previously digested by XmaI and ApaI. All enzymes were purchased from New England Biolabs (Ipswich, MA). Gly to Ser single base mutations were introduced using the QuikChange II kit (Agilent Technologies, Santa Clara, CA). Primers containing single base changes to convert the desired Gly codon, GGC, to a serine codon, AGC, were designed and synthesized (Thermo Fisher Scientific). Mutated plasmids were transformed into XL-1 Blue competent cells. DNA sequencing to confirm fidelity was carried out at Genewiz (South Plainfield, NJ).
3
Cloning and Purification of AlyQ Enzymes
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The full length of AlyQ, excluding its N-terminal signal peptide (C29–Q572), was cloned into the pCold III vector (TaKaRa) between the Nde I and Hind III sites, with a six-His tag inserted before the stop codon. The ligated vector was then transformed into Escherichia coli BL21(DE3) cells and verified by DNA sequencing. The truncated enzymes, AlyQBC, AlyQA, AlyQB and AlyQC, were similarly constructed.
Cells were grown in 1 L of LB broth supplemented with 100 µg/ml ampicillin at 37 °C. The culture was cooled to 15 °C when the OD600 reached 0.6, and isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. After 16-hour incubation, the cells were harvested by centrifugation, resuspended in 50 mM Tris–HCl, 100 mM NaCl, pH 8.0, sonicated and centrifuged again to remove cell debris.
The supernatant was first applied to a Ni2+-charged HiTrap IMAC HP column (GE Healthcare), and eluted with a gradient of 0–1 M imidazole. The protein was then further purified by size-exclusion chromatography using a Superdex 200 column (GE Healthcare). Protein purify was assessed on 10% SDS-PAGE gels. All other truncated enzymes were similarly purified.
Cells were grown in 1 L of LB broth supplemented with 100 µg/ml ampicillin at 37 °C. The culture was cooled to 15 °C when the OD600 reached 0.6, and isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. After 16-hour incubation, the cells were harvested by centrifugation, resuspended in 50 mM Tris–HCl, 100 mM NaCl, pH 8.0, sonicated and centrifuged again to remove cell debris.
The supernatant was first applied to a Ni2+-charged HiTrap IMAC HP column (GE Healthcare), and eluted with a gradient of 0–1 M imidazole. The protein was then further purified by size-exclusion chromatography using a Superdex 200 column (GE Healthcare). Protein purify was assessed on 10% SDS-PAGE gels. All other truncated enzymes were similarly purified.
4
Cloning and Sequencing of DNA Methyltransferase Genes
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To determine the DNA methyltransferase gene responsible for the consecutive methylation in the M. morganii 16S rDNA region (Figure 2B), we selected M.Mom25830ORF6305P and M.Mom25830ORF2065P (designated as M. MmnI in this paper) as candidate genes by searching against a gold-standard dataset in the Restriction Enzyme Database (NEB REBASE) (21) . The genes were cloned into the pCold III-Mor1 expression vector between the SacI (Takara Bio) and XbaI (Takara Bio) restriction sites. For the M.Mom25830ORF6305P-expressing plasmid, the gene-specific oligonucleotide primers used were 5′-GGTGAACGGTTCAGACGACT-3′ and 5′-CCTGCGCTACTGTTTCGGTA-3′ in the first round of nested PCR and 5′-ATATGGAGCTCATGAAAAACACTGTTAATTT-3′ and 5′-TACCTATCTAGATCACGTGAAACTTTCAAGACC-3′ in the second round of PCR. For the M.Mom25830ORF2065P-expressing plasmid (designated pCold III-M. MmnI, Figure 3A), the gene-specific oligonucleotide primers used were 5′-TGTTTTTCCGGCCTTCCTGT-3′ and 5′-CATCGGATTTTCAGCCGCTG-3′ in the first round of nested PCR and 5′-CATATGGAGCTCATGATTTTGAAAAAACACCC-3′ and 5′-TACCTATCTAGATTATTTTACCGGCGGTATTG-3′ in the second round of PCR. The entire cloned gene fragments in both plasmids were Sanger-sequenced.
The pCold III -Mor1 expression vector was constructed from pCold III vector (Takara Bio, Kyoto, Japan) at the NgoMIV
The pCold III -Mor1 expression vector was constructed from pCold III vector (Takara Bio, Kyoto, Japan) at the NgoMIV
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