The PCR conditions were the same as described above Both PCR pro

The PCR conditions were the same as described above. Both PCR products (0.5 μg) were digested with NdeI and HindIII and analyzed by electrophoresis on a 1% agarose gel stained with ethidium bromide. Specifically, approximately 420 bp amplification products were cut out of the gel and purified using the Gel-Out AX kit (A&A Biotechnology, Poland). The purified

DNA fragments were ligated into pET30Ek/LIC between the NdeI and HindIII sites. E. coli strains TOP10F’ cells were transformed with the ligation mixtures and the colonies obtained were examined for the presence of ssb genes from T. maritima and T. neapolitana by PCR amplification and restriction analysis. Single clones, named pETSSBTma and pETSSBTne, were selected and sequenced to ascertain the authenticity of the clones. The constructed plasmids were used in the expression and purification procedure described this website below. Protein sequence analysis of the TmaSSB and TneSSB The amino acid sequences of the TmaSSB and TneSSB proteins were analyzed using standard protein-protein BLAST and RPS-BLAST. Multiple sequence alignments were created using the program MAFFT and the results PD332991 were analyzed and edited using the editor program GeneDoc (copyright by Karl Nicholas). Dendogram of the amino acid sequences of SSB proteins were edited using the editor program Dendroscope [25]. Expression and purification of the TmaSSB and TneSSB The E. coli BL21(DE3)pLysS

strain transformed with pETSSBTma or pETSSBTne was grown at 37°C in 0.5 L LB containing

34 μg/ml kanamycin and 50 μg/ml chloramphenicol to an OD600 of 0.4. Expression was then induced by addition of IPTG to a final concentration of 0.5 mM. After 6 h, the cells were harvested by centrifugation, and suspended in 50 ml buffer A (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA, 0.1% Triton X-100). The purification procedure was very similar to the previously published purification scheme for the SSB from calf thymus [26], and that for thermostable SSB proteins [6]. Generally, the cells were disrupted by sonication and the insoluble debris were removed by centrifugation. The supernatant was heat-treated at 80°C Edoxaban for 20 min and denatured mesophilic proteins were discarded by centrifugation. This supernatant was directly loaded on a QAE-cellulose Selleck KU55933 column (50 ml bed volume, Sigma-Aldrich, USA), from which the proteins were eluted with a linear gradient of 0.05-2 M NaCl in buffer B (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA). The SSB-containing fractions, detected by SDS-PAGE, were combined and loaded on a ssDNA-cellulose column (5 ml, USB, USA). SSB proteins were eluted with gradient of 0.5-1.5 M NaCl and 50% ethylene glycol. The fractions with SSB proteins were collected and dialyzed against buffer B, concentrated using an Amicon Ultra-10 centrifugal filter device (Millipore, USA), and stored at -20°C in buffer C (20 mM Tris-HCl pH 8.

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