glutamicum WT by using primers rbs-ndld and cdld and was cloned i

glutamicum WT by using primers rbs-ndld and cdld and was cloned into the expression vector pEKEx3 [24]. The amplified PCR fragment was ligated to a SmaI bluntend restriction site of pEKEx3. The constructed vector pEKEx3-dld allows the IPTG-inducible expression of dld in C. glutamicum. Because C. efficiens could not be transformed with pEKEx3-dld, dld was amplified using the primer Ex-dld-fw and Ex-dld-bw. The PCR fragment was cloned into the expression vector pVWEx1 [34] via SbfI and KpnI restriction sites. The vector pVWEx1-dld was transformed into C. effiens ZD1839 clinical trial by electroporation

and allowed IPTG-inducible expression of dld in this species. Expression of dld from C. glutamicum ATCC 13032 in Escherichia coli BL21 (DE3) Based on the 5′- and 3′- sequences of dld (accession no. YP_225194) in the genomic DNA of Corynebacterium glutamicum ATCC 13032, the oligonucleotides dld1 and dld2 were designed, and dld was amplified by PCR from the genomic DNA of C. glutamicum ATCC 13032 (1 ng) with dld1and dld2 (0.2 pmol). The thermal profiles for PCR involved the denaturation (94°C for 5 min), 5 cycles of

annealing1 (98°C for 10 sec, 58°C for 30 sec, and 72°C for 90 sec) and subsequently 20 cycles of annealing 2 (98°C for 10 sec, 60°C for 30 sec, 72°C for 90 sec), and the extension (72°C for 7 min). A PCR amplification was carried out with a Blend Taq polymerase in a Gene Amp PCR system 9700 (PE Applied Biosystems, Piscataway, learn more NJ, USA). The resulting 1,020-bp fragment with NdeI and BamHI restriction sites was MX69 sequenced with a DNA sequencing system, SQ5500 (Hitachi, Tokyo,). The obtained dld was ligated into an NdeI and BamHI-digested pT7 Blue-2 T-vector (50 ng/μl) and transformed into E. coli NovaBlue. After cultivation in an LB medium containing ampicillin, the plasmid was extracted with the alkaline mini-prep method and precipitated with polyethylene glycol 6,000. The purified DNA obtained was digested with NdeI and BamHI, and ligated into an NdeI and BamHI-restricted

pET14b vector to form pET14b-dld. pET14b-dld was transformed into E. coli BL21 (DE3). Expression of dld in E. coli BL21 (DE3) and protein purification After the E. coli BL21 (DE3) cells harboring pET14b-dld Decitabine solubility dmso were selected on an LB agar medium containing ampicillin (100 μg/ml), two clones were inoculated into a LB medium (5 ml) containing ampicillin (100 μg/ml) and cultivated at 30°C until the turbidity at 600 nm reached to 0.4-0.8. The culture was inoculated into the same medium (1 l) and cultivated at 30°C for 14 h. The cells were collected by centrifugation (7,100 × g, 10 min), suspended in 0.85% (w/v) NaCl, and centrifuged again. The cells were resuspended in a 20 mM sodium phosphate buffer (pH 8) containing 300 mM NaCl (Buffer A) and stored at -20°C. The cells were disrupted by ultrasonication (model UD-201, Tomy Seiko CO., Tokyo). The disruption conditions used were as follows: output 6; duty cycle 30; and operation time 5 min × 10 times.

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