Pfu DNA polymerase, RNase A, Nde I and Not I, T4 DNA ligase, α-chymotrypsin, pepsin, trypsin, proteinase K and DNA ladder were purchased from Fermentase. Nessler reagent (CAS Number: 7783-33-7), kanamycin (K1377–5G), agarose (A9539–50G) ANS (A 1028) were provided from sigma Aldrich Co (Steinheim, USA). Anti poly-Histidine-HRP antibody was purchased from sigma (A7058). Protein molecular mass marker, IPTG, amino acids, DTT, BME, GSH, ascorbic acid, iodoacetate, iodoacetamide were acquired from Merck. DNA extraction kit was purchased from Peqlab and PCR product purification kit was obtained from BioNEER (Seoul, Korea). Ni-NTA resin was purchased from Invitrogen (Carlsbad, USA). E. coli DH5α and BL21 (DE3) strains and pET-26b vector resistant to kanamycin were purchased from Invitrogen (Carlsbad, USA). All experiments were replicated at least three times.
Cultivation of Cohnella sp. A01, was carried out at 55 °C in LB medium for 4 days and it΄s genomic DNA was extracted using the high pure DNA extraction kit. E. coli strains containing pET-26b vector were cultured at 37 °C in LB with final concentration 30 μg/ml of kanamycin. LB plates were solidified with 1.5% agar in the presence and absence of kanamycin to perform respectively contamination and viability tests.
Cultivation of Cohnella sp. A01, was carried out at 55 °C in LB medium for 4 days and it΄s genomic DNA was extracted using the high pure DNA extraction kit. E. coli strains containing pET-26b vector were cultured at 37 °C in LB with final concentration 30 μg/ml of kanamycin. LB plates were solidified with 1.5% agar in the presence and absence of kanamycin to perform respectively contamination and viability tests.